Reducing disease transmission of vector-borne diseases

Abstract

This application relates to compounds and pharmaceutically acceptable salts thereof, compositions thereof and their use in reducing or preventing the transmission of parasitic or vector-borne diseases.

Description

[Technical Field]

[0001] This 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 technology]

[0002] Infectious diseases are the cause of a wide variety of illnesses of medical and veterinary importance. Many of these diseases are transmitted by insect vectors. Vector-borne diseases are infections transmitted by the bite of infected arthropod species such as mosquitoes, ticks, triatomine bugs, sandflies, black flies, and ectoparasites such as mites and fleas.

[0003] Mosquitoes are vectors of various infectious diseases. Three medically relevant genera of mosquitoes that transmit diseases are Anopheles, Culex, and Aedes. Culex and Aedes belong to the subfamily Culicinae, while Anopheles belongs to the subfamily Anophelinae. 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, and filariasis.

[0004] Filariasis is a disease caused by nematode parasites of the filarioidea type and is spread by blood-sucking insects such as blackflies and mosquitoes. They are divided into categories based on the location of their effect. Lymphatic filariasis is caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, which occupy the lymphatic system and can result in elephantiasis. Subcutaneous filariasis is caused by Loa (eyeworm), Mansonella streptocerca, and Onchocerca volvulus, which occupy the layer just below the skin. Serous filariasis is caused by the helminths Mansonella perstans and Mansonella ozzardi, which occupy the serous cavities of the abdomen. Circulating microfilariae can be picked up by insect vectors during blood meals, where they develop into infective larvae that can be spread to other people.

[0005] Zika virus disease is caused by members of the Flaviviridae family of viruses. It is spread by day-active Aedes mosquitoes, such as Aedes aegypti and Aedes albopictus. Symptoms include fever, bloodshot eyes, joint pain, headache, and a maculopapular rash. Most cases are asymptomatic, but when they do appear, they are usually mild, may resemble dengue fever, and generally last less than seven days. Infection during pregnancy can cause microcephaly and other brain abnormalities 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 transmitted primarily by Culex species of mosquitoes. Approximately 80% of infected individuals experience few or no symptoms. 20% of infected individuals develop fever, headache, vomiting, or a rash. Fewer than 1% develop encephalitis or meningitis with associated neck stiffness, confusion, or seizures, with a case-fatality rate of approximately 10%. Recovery can take weeks to months, and individuals with affected nervous systems have a 10% risk of death.

[0007] Chikungunya virus is a member of the Alphavirus genus and the Togaviridae family. It is an RNA virus with an 11.7 kb positive-sense, single-stranded genome. Chikungunya virus causes chikungunya, a disease that typically manifests as fever and joint pain 2 to 12 days after exposure. The risk of death is approximately 1 in 1,000. The disease causes an estimated 3 million infections each year. Chikungunya is mostly found in developing countries, but outbreaks in the Indian Ocean, Pacific islands, and the Americas continue to shift the disease's distribution.

[0008] Mayaro virus disease is caused by members of the Togaviridae family, specifically the Alphavirus genus. Symptoms include fever, headache, myalgia, rash, prominent pain in large joints, and association with rheumatic disease. It is known to be widespread in South America. Transmission of MAYV is primarily maintained by a sylvatic cycle involving nonhuman primates and Haemagogus mosquitoes.

[0009] Sandflies 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 is potentially fatal if untreated. Leishmaniasis is widespread in developing countries; approximately 90 percent of the world's cases of visceral leishmaniasis are in India, Bangladesh, Nepal, Sudan, and Brazil. Leishmaniasis affects 12 million people worldwide, with 1.5 to 2 million new cases each year. Visceral leishmaniasis has an estimated incidence of 500,000 new cases and 60,000 deaths each year. Kabul is estimated to be the world's largest center of cutaneous leishmaniasis, with approximately 67,500 cases as of 2004.

[0010] Kissing bugs, also known as conenose bugs, are members of the Triatominae subfamily of the Reduviidae family. They are vectors of Chagas disease, also known as American trypanosomiasis. Chagas disease is a parasitic disease caused by the flagellated protozoan Trypanosoma cruzi. Chagas disease is generally widespread in the Americas and is endemic to poor, remote areas of Mexico, Central America, and South America. An estimated 10 to 15 million people are infected with Chagas disease annually, resulting in approximately 14,000 deaths annually. Symptoms of Chagas disease vary over the course of an infection. In the early (acute) phase, symptoms are mild, typically localized swelling at the site of infection. After 4 to 8 weeks, individuals with active infection enter the chronic phase of Chagas disease, during which 60 to 80 percent of chronically infected individuals remain asymptomatic throughout their lifetime. However, the remaining 20 to 40 percent of infected people will develop debilitating and potentially life-threatening medical problems over the course of their lives. Chagas disease is treated with nifurtimox and benznidazole, which cause significant side effects and offer little benefit in chronic cases.

[0011] Tsetse flies (Glossina spp.) are vectors of human African trypanosomiasis. Human African trypanosomiasis, also known as African sleeping sickness, is a parasitic disease caused by the protozoan Trypanosoma brucei. Two forms of the disease exist, depending on the subspecies of the parasite. Trypanosoma brucei gambiense (TBgambiense) accounts for 95% of reported cases and is present in West and Central Africa, where it causes a chronic infection. Trypanosoma brucei rhodesiense (TBrhodesiense) is found in East and Southern Africa, where it accounts for approximately 5% of cases.

[0012] These diseases are of great medical importance. Several 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 dengue virus. Furthermore, in the case of antimalarial drugs, treatment with currently available drugs has become less effective due to increasing resistance in some Plasmodium strains. Therefore, there is a need to effectively control parasites and disease vectors to prevent transmission. In this regard, mosquitoes can be targeted with a wide variety of insecticides and insect repellents. Mosquitoes can be targeted with insecticides when they are in their larval state or when they become adults. However, mosquitoes have developed widespread resistance to currently used insecticides. Summary of the Invention

[0013] One approach to addressing this problem is to develop drugs that can reduce or prevent the transmission of vector-borne diseases without negatively affecting the insect vector, thereby avoiding the development of 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 malaria in mosquitoes. Furthermore, there is an ongoing need to develop novel modalities for reducing or preventing the transmission of vector-borne diseases.

[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, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising 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 parasite transmission, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, the method comprising 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 the transmission of a vector-borne disease or parasitic infection, the method comprising 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 the 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 for use in reducing or preventing vector-borne or parasitic diseases.

[0020] In any of the above-listed embodiments, the vector-borne or parasitic disease may be selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii 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 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 several respects. In particular, the present inventors have 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 are responsible for preventing the transmission of vector-borne diseases and parasites in vectors (e.g., mosquitoes, sandflies, tsetse flies, triatominae, etc.). In some embodiments, 1-methyl-9H-pyrido[3,4-b]indole, harman, or 1-methyl-β-carboline is produced by bacteria of the genus Delftia. When introduced into a vector-containing environment, the compositions of the present invention prevent the development of parasites in the vector, thus disrupting disease transmission. 1-methyl-9H-pyrido[3,4-b]indole may 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, the method comprising the step of contacting at least one vector with the composition such that the vector orally ingests the composition, and wherein the vector-borne disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii disease, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, Ehrlichia muris-like disease, ehrlichiosis, filariasis, Heartland virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific 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 present invention, there is provided a composition for use in a method for reducing or preventing parasitic transmission, comprising a bacterium of the genus Delftia, wherein the composition comprises a bacterium of the genus Delftia, the method comprising the step of contacting at least one vector with the composition such that the vector orally ingests the composition, and the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Borrelia mayonii disease, Chagas disease, Cryptosporidium, dirofilariasis, Ehrlichia muris-like disease, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific tick fever, Rocky Mountain spotted fever, and tularemia.

[0024] In a further aspect of the invention, there is provided a bacterium of the genus Delftia for use in reducing or preventing the transmission of a disease selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii 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 tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0025] Throughout this document, bacteria of the genus Delftia may properly be referred to as Delftia tsuruhatensis TC1.

[0026] The invention will be further described by reference to the accompanying non-limiting drawings in which: [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows a liquid chromatography-high resolution accurate mass spectrometer trace for the active ingredient of Delftia tsuruhatensis. [Figure 2] FIG. 1 shows heteronuclear single quantum coherence spectroscopy of the active ingredient of Delftia tsuruhatensis. [Figure 3] FIG. 1 shows the H NMR spectrum of the active ingredient of Delftia tsuruhatensis. [Figure 4] FIG. 1 shows the effect of Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 5A]FIG. 1 shows the effect of timing of (i) feeding and (ii) exposure to Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 5B] FIG. 1 shows the effect of timing of (i) feeding and (ii) exposure to Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 5C] FIG. 1 shows the effect of timing of (i) feeding and (ii) exposure to Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 6] FIG. 1 shows the effect of different blood meals on L. major parasites in sandflies fed Delftia tsuruhatensis. [Figure 7] FIG. 1 shows the effect of Delftia tsuruhatensis against sandflies naturally infected by bites on infected skin lesions in mice. [Figure 8A] FIG. 1 shows the effect of exposure to Delftia tsuruhatensis on sandfly mortality. [Figure 8B] FIG. 1 shows the effect of exposure to Delftia tsuruhatensis on sandfly mortality. [Figure 8C] FIG. 1 shows the effect of exposure to Delftia tsuruhatensis on sandfly mortality. [Figure 9A] FIG. 1 shows the effect of E. coli or other bacteria such as Ornithinibacillus massiliensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 9B] FIG. 1 shows the effect of dead Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. [Figure 10A]FIG. 1 shows the effect of exposing sandflies to Delftia tsuruhatensis on the transmission of leishmania to mice. [Figure 10B] FIG. 1 shows the effect of exposing sandflies to Delftia tsuruhatensis on the transmission of leishmania to mice. [Figure 10C] FIG. 1 shows the effect of exposing sandflies to Delftia tsuruhatensis on the transmission of leishmania to mice. [Figure 11] FIG. 1 shows the effect of harman on Zika virus reproduction in mosquitoes. DETAILED DESCRIPTION OF THE INVENTION

[0028] In one aspect, the invention provides a composition for use in a method for reducing or preventing vector-borne disease transmission, 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 the method comprises contacting at least one vector with the composition.

[0029] 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.

[0030] As used herein, the term "vector-borne zoonotic disease" refers to a disease that naturally infects wildlife and is then transmitted to humans by carriers or vectors such as mosquitoes, ticks, tsetse flies, black flies, Triatominae, and sand flies. As used herein, the term "vector-borne disease" refers to a disease that naturally infects wildlife and is then transmitted to animals by carriers or vectors such as 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 the group consisting of a dog, cat, hamster, cow, sheep, goat, pig, rabbit, duck, turkey, horse, chicken, yak, donkey, water buffalo, camel, or other livestock.

[0031] Delftia is a genus of gram-negative motile rod-like bacteria belonging to the class Betaproteobacteria and family Comamonadaceae.

[0032] The Delftia bacterium may be any bacterium of the genus Delftia. In one embodiment of the present invention, the Delftia bacterium is D. tsuruhatensis. The bacterial strain TC1 was deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland) on May 21, 2019, under the accession number NCIMB43398. This bacterium was isolated and identified as D. tsuruhatensis 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 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) on May 21, 2019, under accession number NCIMB43398, and the whole genome sequence was deposited at NCBI (National Center for Biotechnological Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, United States) on October 14, 2022, under accession number PRJNA890603.

[0033] 1-Methyl-9H-pyrido[3,4-b]indole, also known as harman, has the following structure: [ka] It is expressed by:

[0034] This compound has been found to be an active compound secreted by Delftia bacteria and can inhibit the transmission of parasites in various vectors. Thus, the compositions of the present invention can reduce or prevent disease or parasite transmission in mosquitoes.

[0035] It will further be understood that 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 isomeric forms depends on the environment in which the compound is present.

[0036] The compound may also be protonated or deprotonated depending on the pH of its surrounding environment.The compound may also be in the form of a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include but are not limited to those listed in Berge, J.Pharm.Sci., 1977, 66, 1-19, or those listed in PH Stahl and CG Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Edition, John Wiley & Sons, March 2011.

[0037] Where the functionality of the compound allows, suitable pharmaceutically acceptable salts of the compound of formula (I), including acid or base addition salts, may be formed. Acid addition salts may be formed by reaction with an appropriate acid, optionally in a suitable solvent such as an organic solvent, to obtain a salt that can be isolated by crystallization and filtration. Base addition salts may be formed by reaction with an appropriate base, optionally in a suitable solvent such as an organic solvent, to obtain a salt that can be isolated by crystallization and filtration.

[0038] Representative pharmaceutically acceptable acid addition salts include 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecyl benzoate, and benzoic acid salts. Ethylenediaminetetraacetate (EDTA), Lauryl Sulfate (Estolate), Ethane-1,2-Disulfonate (Edisylate), Ethanesulfonate (Esylate), Formate, Fumarate, Galactarate (Mucate), Gentisate (2,5-Dihydroxybenzoate), Glucoheptonate (Gluceptate), Gluconate, Glucuronate, Glutamate, Glutarate, Glycerophosphate, Glycolate, Hexylresorcinate, Hippurate, Hydrabamine (N,N'-di(dehydroacetate) (biethyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methyl sulfate, mucate, naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicyclate, pamoate (embonate) ), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophylline), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate salts.

[0039] Representative pharmaceutically acceptable base addition salts include aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolizidine-1'-ylmethylbenzimidazole), cyclobenzylamine, cyclohexylmethylbenzoimidazole ... These include, but are not limited to, hexylamine, 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, f-butylamine, and zinc.

[0040] The compounds are administered in an appropriate "effective amount," which will depend on several factors, including, for example, the size and weight of the subject, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of delivery, and is ultimately at the discretion of one of ordinary skill in the art.

[0041] In some embodiments, the vector-borne disease is selected from African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Bourbon virus, Borrelia mayonii disease, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, Ehrlichia muris-like infection, ehrlichiosis, filariasis, Heartland virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0042] In some embodiments, the composition comprises a bacterium of the genus Delftia, and the vector-borne disease is selected from African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Bourbon virus, Borrelia mayonii disease, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, Ehrlichia muris-like infection, ehrlichiosis, filariasis, Heartland virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0043] 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 African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Bourbon virus, Borrelia mayonii disease, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, Ehrlichia muris-like infection, ehrlichiosis, filariasis, Heartland virus, Japanese encephalitis, Lyme disease, Mayaro virus disease, Pacific tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0044] In a second aspect, the present invention provides a composition for use in a method for reducing or preventing parasitic transmission, the composition comprising a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising contacting at least one vector with the composition.

[0045] In some embodiments, the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Borrelia mayonii disease, Chagas disease, Cryptosporidium, dirofilariasis, Ehrlichia muris-like infections, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific tick fever, Rocky Mountain spotted fever, and tularemia.

[0046] In some embodiments, the composition for use in the method of reducing or preventing parasitic transmission comprises a bacterium of the genus Delftia, and the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Borrelia mayonii disease, Chagas disease, Cryptosporidium, dirofilariasis, Ehrlichia muris-like infections, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific tick fever, Rocky Mountain spotted fever, and tularemia.

[0047] In some embodiments, a composition for use in a method for 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 disease, Borrelia mayonii disease, Chagas disease, Cryptosporidium, Dirofilariasis, Ehrlichia muris-like infection, ehrlichiosis, filariasis, Lyme disease, Pacific tick fever, Rocky Mountain spotted fever, and tularemia.

[0048] In particular, harman has been shown herein to be capable of preventing parasite transmission when introduced into a vector-containing environment. In some embodiments, the mode of introduction into the vector may be via contact with sugar prey, nectar prey, blood prey, and / or other food sources, whereby Delftia bacteria and / or 1-methyl-9H-pyrido[3,4-b]indole may be delivered to and / or within the vector via cuticular uptake and / or ingestion. Thus, the compositions of the present invention can reduce or prevent disease and / or parasite transmission in the vector. The vector may be any vector capable of transmitting disease, and in some embodiments, may be a mosquito, such as a mosquito of the genus Anopheles. It is contemplated that the compositions and methods of the present invention extend to any Anopheles species of mosquito. In certain embodiments, the mosquito is Anopholes gambiae, Anopholes stephensi, Anopholes culicifacies, or Anopholes coluzzi. In one embodiment of the invention, the mosquito is Anopheles gambiae or Anopheles stephensi. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0049] In some embodiments, the composition is for use in reducing or preventing (i) Zika virus and / or (ii) Zika virus transmission. In some embodiments, the composition is for use in reducing or preventing (i) Zika virus and / or (ii) Zika virus transmission in mosquitoes. In some embodiments, the composition is for use in reducing or preventing Zika virus. In some embodiments, the composition is for use in reducing or preventing Zika virus transmission in mosquitoes. The mosquito may be any mosquito. In some embodiments, the mosquito is of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0050] In some embodiments, the composition is for use in reducing or preventing the transmission of (i) chikungunya virus and / or (ii) chikungunya virus. In some embodiments, the composition is for use in reducing or preventing the transmission of (i) chikungunya virus and / or (ii) chikungunya virus in mosquitoes. In some embodiments, the composition is for use in reducing or preventing chikungunya virus. In some embodiments, the composition is for use in reducing or preventing the transmission of chikungunya virus in mosquitoes. The mosquito may be any mosquito. In some embodiments, the mosquito is of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0051] In some embodiments, the composition is for use in reducing or preventing (i) filariasis or dirofilariasis, and / or (ii) the transmission of filariasis or dirofilariasis. In some embodiments, the composition is for use in reducing or preventing (i) filariasis or dirofilariasis, and / or (ii) the transmission of filariasis or dirofilariasis in mosquitoes. In some embodiments, the composition is for use in reducing or preventing filariasis or dirofilariasis. In some embodiments, the composition is for use in reducing or preventing filariasis or dirofilariasis. In some embodiments, the composition is for use in reducing or preventing the transmission of filariasis or dirofilariasis in mosquitoes. The mosquito may be any mosquito. In some embodiments, the mosquito is of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0052] In some embodiments, the composition is for use in reducing or preventing the transmission of (i) Mayaro virus and / or (ii) Mayaro virus. In some embodiments, the composition is for use in reducing or preventing the transmission of (i) Mayaro virus and / or (ii) Mayaro virus in mosquitoes. In some embodiments, the composition is for use in reducing or preventing the transmission of Mayaro virus. In some embodiments, the composition is for use in reducing or preventing the transmission of Mayaro virus in mosquitoes. The mosquito may be any mosquito. In some embodiments, the mosquito is of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0053] In some embodiments, the composition is for use in reducing or preventing (i) West Nile Virus and / or (ii) West Nile Virus transmission. In some embodiments, the composition is for use in reducing or preventing (i) West Nile Virus and / or (ii) West Nile Virus transmission in mosquitoes. In some embodiments, the composition is for use in reducing or preventing West Nile Virus transmission in mosquitoes. The mosquito may be any mosquito. In some embodiments, the mosquito is of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0054] In some embodiments, the composition is for use in reducing or preventing (i) Chagas disease and / or (ii) transmission of Chagas disease. In some embodiments, the composition is for use in reducing or preventing transmission of Chagas disease in Triatominae. In some embodiments, the composition is for use in reducing or preventing transmission of the Chagas disease parasite in Triatominae. In some embodiments, the Triatominae is Triatoma dimidiate.

[0055] In some embodiments, the composition is for use in reducing or preventing the transmission of (i) human African trypanosomiasis and / or (ii) human African trypanosomiasis. In some embodiments, the composition is for use in reducing or preventing the transmission of (i) human African trypanosomiasis and / or (ii) human African trypanosomiasis in tsetse flies. In some embodiments, the composition is for use in reducing or preventing human African trypanosomiasis. In some embodiments, the composition is for use in reducing or preventing the transmission of human African trypanosomiasis in tsetse flies. In some embodiments, the tsetse fly is of the genus Glossina. In some embodiments, the tsetse fly is Glossina palpalis.

[0056] In some embodiments, the composition is for use in reducing or preventing the transmission of (i) leishmaniasis and / or (ii) leishmaniasis. In some embodiments, the composition is for use in reducing or preventing the transmission of (i) leishmaniasis and / or (ii) leishmaniasis in sandflies. In some embodiments, the composition is for use in reducing or preventing leishmaniasis. In some embodiments, the composition is for use in reducing or preventing the transmission of leishmaniasis in sandflies. In some embodiments, the sandflies are of the genus Phlebotomus or Lutzomyia.

[0057] In some embodiments, the composition comprises a bacterium of the genus Delftia and is for use in reducing or preventing the transmission of (i) leishmaniasis and / or (ii) leishmaniasis. In some embodiments, the composition is for use in reducing or preventing the transmission of (i) leishmaniasis and / or (ii) leishmaniasis in sandflies. In some embodiments, the composition is for use in reducing or preventing leishmaniasis. In some embodiments, the composition is for use in reducing or preventing the transmission of leishmaniasis in sandflies. In some embodiments, the sandflies are of the genus Phlebotomus or Lutzomyia.

[0058] In some embodiments, the composition is for use in reducing or preventing (i) Cryptosporidium and / or (ii) Cryptosporidium transmission. In some embodiments, the composition is for use in reducing or preventing Cryptosporidium transmission. In some embodiments, the composition is for use in reducing or preventing Cryptosporidium transmission.

[0059] In another aspect, the present invention provides a method for reducing or preventing the transmission of a vector-borne disease or parasite, comprising contacting at least one vector of the vector-borne disease or parasite 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 parasite may be as defined above.

[0060] The step of contacting the vector or parasite with the composition can be carried out in any suitable manner. For example, it is not necessary for a person to physically contact the vector or parasite with the Delftia bacterium and / or 1-methyl-9H-pyrido[3,4-b]indole or its pharmaceutically acceptable salt. The composition can be left in a location where it will contact the vector. The composition can be in the form described above or below.

[0061] In certain embodiments of the invention, contacting may be accomplished by treating the area with a composition of the invention, e.g., by using a spray formulation such as an aerosol or pump spray. In certain embodiments of the invention, the area may be treated, e.g., by airdrop, with truck-mounted equipment, etc. In some embodiments, the composition is sprayed, e.g., by backpack spraying, aerial spraying, spraying / dusting, etc. The vector or parasite may be any vector of a parasite capable of transmitting disease. In some embodiments, the vector is a mosquito, blackfly, triatominae, sandfly, or tsetse fly.

[0062] 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 Anopholes. In some embodiments, the mosquito is Anopholes gambiae, Anopholes stephensi, Anopholes culicifacies, or Anopholes 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.

[0063] For filariasis, the method includes contacting a mosquito or blackfly with 9H-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The mosquito may 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.

[0064] In some embodiments, the dirofilariasis is caused by Dirofilaria immitis, Dirofilaria repens, or Dirofilaria tenuis. In some embodiments, the dirofilariasis is caused by Dirofilaria immitis. In some embodiments, the dirofilariasis is caused by Dirofilaria repens. In some embodiments, the dirofilariasis is caused by Dirofilaria tenuis.

[0065] In some embodiments, the filariasis is caused by a worm of the genus Filarioidea. In some embodiments, the worm is Wuchereria bancrofti, Brugia malayi, or Brugia timori. In some embodiments, the worm is Loa loa, Mansonella streptocerca, or Onchocerca volvulus. In some embodiments, the worm is Mansonella perstans or Mansonella ozzardi.

[0066] For Chagas disease, the method includes contacting infected Triatominae with a composition of the present invention. The Triatominae may be any Triatominae capable of transmitting Chagas disease, such as Triatoma infestans, Triatoma dimidiate, or Rhodnius prolixus. In some embodiments, the Triatominae is Triatoma infestans. In some embodiments, the Triatominae is Triatoma dimidiate. In some embodiments, the Triatominae is Rhodnius prolixus. The parasite may be any parasite that causes Chagas disease. In some embodiments, the parasite is Trypanosoma cruzi.

[0067] For leishmaniasis, the method comprises contacting an infected sandfly with a composition of the present invention. In some embodiments, the method comprises contacting an infected sandfly with a composition comprising a bacterium of the genus Delftia. The sandfly may be any sandfly capable of transmitting Leishmania. It is contemplated that the compositions and methods of the present invention extend to any species of sandfly. In some embodiments, the sandfly is of the genus Phlebotomus or Lutzomyia.

[0068] The parasite may be any leishmaniasis parasite. In some embodiments, the parasite is Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania panamensis, Leishmania guayanensis, Leishmania amazonensis, Leishmania mexicana, Leishmania tropica, or Leishmania major. In some embodiments, the parasite is Leishmania donovani. In some embodiments, the parasite is Leishmania infantum. In some embodiments, the disease being treated is visceral leishmaniasis. In other embodiments, the disease being treated is cutaneous leishmaniasis.

[0069] For human African trypanosomiasis, the method includes contacting an infected tsetse fly with a composition of the present invention. The tsetse fly may be any tsetse fly capable of transmitting human African trypanosomiasis. It is contemplated that the compositions and methods of the present invention extend to any species of tsetse fly. In some embodiments, the tsetse fly is Glossina palpalis.

[0070] The parasite may be any parasite that causes human African trypanosomiasis. 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).

[0071] For Cryptosporidium, the method includes contacting the Cryptosporidium with a composition of the present invention. Cryptosporidium is a parasitic alveolate of the Apicomplexan that causes respiratory and gastrointestinal disease. The parasite may be any parasite that causes Cryptosporidium, and the compositions and methods of the present invention are contemplated to cover any Cryptosporidium parasite. In some embodiments, the parasite is selected from Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium meleagridid, and Cryptosporidium muris.

[0072] 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.

[0073] composition The compositions of the present invention may be in any suitable form and may include any suitable carrier. The composition may be a feed composition, i.e., the composition may be in a form that can be provided to the vector for oral consumption. In some embodiments, 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 may be included in the attractive sugar bait. The attractive sugar bait comprises a sugar and a toxic component. It is envisioned that the attractive sugar bait of the present invention comprises the composition of the present invention instead of the toxic component, i.e., comprises a sugar and the composition of the present invention. In some embodiments, the composition is in the form of a bait. The bait is designed to lure the vector (e.g., mosquito) to come into contact with the composition. In some embodiments, upon contact, the composition is then taken up by the vector or parasite (e.g., mosquito) by, for example, ingestion. An attractant may also be used. The attractant may be a pheromone, such as a male or female pheromone. The attractant acts to lure the vector (e.g., mosquito) to the bait. The bait may be in any suitable form such as a solid, paste, pellet or powder form.

[0074] The bait may be provided in a suitable "housing" or "trap." Such housings and traps are commercially available, and existing traps can be adapted to contain the compositions of the present invention. The housing or trap may be, for example, box-shaped, provided pre-formed, or formed, for example, from foldable cardboard. Suitable materials for the housing or trap include plastic and cardboard, particularly corrugated cardboard. The inner surface of the trap may be lined with an adhesive substance to restrict the movement of the vector or parasite (e.g., mosquito) once inside the trap. The housing or trap may contain a suitable bait container that can hold the bait in place. Traps are distinguished from housings because the mosquitoes cannot easily exit the trap after entry, but the housing acts as a "feeding station" that provides the vector (e.g., mosquito) with a favorable environment where they can eat and feel safe from predators.

[0075] In another embodiment, the present invention provides a nectar diet for mosquitoes, comprising one or more of a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and a sugar source.

[0076] In a further embodiment of the invention, bacteria of the genus Delftia may be used in disease control strategies based on direct exposure of the larval or adult stage of the vector to the bacteria, which may be achieved by direct administration of the bacteria.

[0077] Alternatively, the bacteria may be placed directly into the local vector population.

[0078] The bacteria may be delivered in combination with a delivery agent by any suitable method and in any suitable manner that allows the composition to be administered to the vector, for example, the vector may be contacted with a solution containing the bacteria, e.g., Delftia, in pure or substantially pure form.

[0079] In certain embodiments, the Delftia bacteria is in a composition in combination with a delivery agent.

[0080] In another specific embodiment, the larval form of the vector (eg, a mosquito) may simply be "dipped" or "sprayed" with a solution containing the bacteria.

[0081] combination It is envisioned that the present invention will be utilized in conjunction with other anti-vector or anti-parasite eradication efforts. For example, the compositions, methods, and compounds for use of the present invention may be used together with known anti-vector or anti-parasite agents (such as antimalarials). In some embodiments, the compositions or compounds for use of the present invention may be used in combination with one, two, or three additional anti-parasite agents (e.g., antimalarials). Integrated Vector Management (IVM) proposes making full use of available tools.

[0082] The at least one other antimalarial agent may also be selected from ferroquine, KAF156, sipargamine, DSM265, artemisone, artemisinone, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, OZ609, OZ277, and SAR97276. In treating P. falciparum infection, the at least one, two, or three additional antimalarial agents may be selected from the following list, wherein at least one of the antimalarials 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 treatments are known as artemisinin-based combination therapies (ACTs). The selection of an ACT is usually based on the results of therapeutic efficacy studies against local strains of P. falciparum malaria. In the treatment of P. vivax infections, ACTs may be used as described above. Alternatively, the at least one other antimalarial agent may be chloroquine, particularly in areas where chloroquine-resistant P. vivax is not present. In areas where resistant P. vivax is confirmed, the infection may be treated with ACTs as described above. The combination of therapeutic agents may be conveniently provided for use in the form of a pharmaceutical composition or formulation, and may be administered together or separately, and if administered separately, this may be done separately or sequentially in any order (by the same or different routes of administration).

[0083] The compositions or bacteria for use in the present invention may be used with insecticide-soaked mosquito nets (ITNs), including long-lasting mosquito nets (LLINs), and / or indoor residual sprays (IRS). The ATSB (attractive and toxic sugar bait) lures mosquitoes into eating the sugar along with a toxic mosquito-killing compound. For efficiency, the ATSB can also contain the harmane compound discussed above instead of the toxic compound. [Example]

[0084] The present invention will now be illustrated by the following non-limiting examples. While specific embodiments of the present invention are described below, those skilled in the art will understand that various changes and modifications may be made. Reference to preparations made in a manner similar to other preparations, or by their general methodology, can encompass variations of routine parameters, such as minor changes in time, temperature, workup conditions, and amounts of reagents.

[0085] Supernatant preparation D. tsuruhatensis TC1 was grown overnight in LB liquid medium (200 rpm, 28°C). The bacteria were washed and resuspended in M9 medium (10 9 The supernatant was filtered through a 0.22 μm filter to generate D. tsuruhatensis TC1 (D-8h). The supernatant was then passed through a 3 kDa centrifugal filter (Amicon Ultra-3K, REF: UFC500396) to generate a <3 kDa fraction.

[0086] Example 1: Bioassay-guided purification of active natural components from the supernatant of Delftia tsuruhatensis TC1 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.

[0087] Material not retained in the column (the flow-through was collected for activity testing) and the column was eluted at 10 mL / min with an isocratic elution (HO / CHCN 95:5), followed by a gradient of 5% to 100% acetonitrile (CHCN) in water in 40 min and an isocratic step of 100% CHCN in 20 min. Fractions of 18 mL were collected. UV detection at 210 nm and 280 nm was used.

[0088] Additionally, 100 mL of non-fermented medium was loaded onto a 60 x 15 mm C-18 cartridge and eluted with 100 mL of 100% CH3CN, which was dried (12.5 mg) and dissolved in 1 mL of MeOH.

[0089] 500 μL of the supernatant, 500 μL aliquots of each fraction, 500 μL of the follow-through obtained while loading 10 L of the supernatant onto the C-18 column, and 100 μL of blank medium extract were transferred to an AB-Gene0765 800 μL 96-well storage plate and dried in an HT-8 Genevac vacuum centrifuge for transport and activity assessment.

[0090] LC-HRMS de-iteration method Active fractions were analyzed using an Agilent 1200 Rapid Resolution HPLC coupled to a Bruker maXis mass spectrometer. The applied sample volume was 2 μL. A Zorbax SB-C8 column (2.1 × 30 mm, 3.5 μm particle size) was used for the separation. Two solvents were used as mobile phases: Solvent A HO:CHCN 90:10, Solvent B water:CHCN 10:90, both with 13 mM ammonium formate and 0.01% TFA. The gradient composition was as follows: [Table 1]

[0091] The mass spectrometer was operated in positive ESI mode. Instrument parameters were 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. TFA-Na cluster ions were used for instrument mass calibration before sample injection. Each sample run was recalibrated by injection of the same TFA-NA calibrant before the start of chromatography.

[0092] NMR de-iteration method For NMR analysis, samples were dissolved in CD3OD. After dissolution, each sample was transferred to a 1.7 mm tube. 1H spectra and 2D HSQC spectra) were performed on a Bruker AVANCE III 500 MHz spectrometer equipped with a 1.7 mm TCI microcryoprobe. All spectra were registered at 24 °C.

[0093] result C 12 H 10 The molecular formula of N2 was determined from LC-HRMS shown in Figure 1 for the active ingredient.

[0094] HSQC of active ingredients and 1 The 1 H NMR spectrum was consistent with that of harman, as shown in Figures 2 and 3.

[0095] Example 2: Effect of Delftia tsuruhatensis on the establishment of leishmania parasites in the midgut of sandflies. Materials and methods used to establish the role of Delftia TC1 in interfering with leishmania infection in sandflies parasites A clonal strain of Leishmania major (WR2885) was used [Cecilio 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.]

[0096] Promastigotes were maintained at 26°C in Schneider's insect medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin (all Thermo Fischer Scientific).

[0097] mouse Six-week-old female BALB / c mice were obtained from Charles River laboratories and housed under pathogen-free conditions at the NIAID Twinbrook Animal Facility (Rockville, MD) with water and food ad libitum.

[0098] Sandflies Phlebotomus duboscqui sand flies were mass-reared in the Laboratory of Malaria and Vector Research insectary 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.

[0099] Adult females were maintained on a diet of 30% sucrose and starved for 12 h before feeding.

[0100] Data Representation and Statistics Results from at least two independent experiments are presented for each individual sandfly / mouse as group mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software v6.01. The nonparametric Mann-Whitney test was used to assess statistical differences with a p < .05.

[0101] Figure 4: Effect of Leishmania major parasite establishment in sandflies fed Delftia tsuruhatensis TC1 bacteria P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of Delftia tsuruhatensis TC1 bacteria (1 × 10 in 5% sucrose solution). 8 CFU / ml) for 24 hours (denoted as "Bacteria" in the figure), while a control group of sandflies (denoted as "Control" in the figure) was fed 5% sucrose alone.

[0102] After an overnight starvation period, sandflies were challenged with L. major promastigotes (5 × 10) as described by Cecilio 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. 6 Chicks were infected by artificial feeding through the membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml.

[0103] After infection, blood-fed females were selected and maintained on a 30% sucrose diet. Seven (and / or eight) and eleven days after infection, sandflies were collected to determine infection status. Briefly, under a stereomicroscope, sandfly midguts were dissected in PBS and transferred to individual microtubes (Denville Scientific) containing 50 μL of formalin solution (0.005% in PBS). The midguts were homogenized, and 10 μL was loaded into a disposable Neubauer chamber (Incyto). Slides were observed under a phase-contrast microscope (Zeiss) at 400x magnification.

[0104] Total parasite counts and frequency of developmental terminals [Cecilio 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.] were determined.

[0105] Data are presented in the form of dot plots, with each symbol corresponding to a single midgut. Statistical differences were determined using the nonparametric Mann-Whitney test, with a value of at least P ≤ .05 indicating statistical relevance.

[0106] The data shown in Figure 4 (A, B, and C) indicate that exposure of sandflies to Delftia tsuruhatensis TC1 affects the establishment of Leishmania parasites in the sandfly midgut. It can be seen that parasite levels are significantly reduced in sandflies exposed to the bacteria compared to sandflies without Delftia tsuruhatensis TC1 bacteria. Leishmania infection in sandflies exposed to Delftia tsuruhatensis TC1 showed a significant reduction in parasite burden (p<0.0001) (Figure 4C) compared to Delftia tsuruhatensis TC1-treated sandflies at 7 days (p<0.5) (Figure 4A) or 8 days (p<0.01) (Figure 4B) after Leishmania infection.

[0107] The data shown in Figure 4 (D, E, F, and G) indicate that exposure of sandflies to Delftia tsuruhatensis TC1 does not affect the percentage of metacyclic parasites, but the total number of parasites is significantly reduced in the midgut of sandflies exposed to the bacteria compared to sandflies without Delftia tsuruhatensis TC1 bacteria.

[0108] Figure 5: Effect of timing of (i) feeding and (ii) exposure to Delftia tsuruhatensis TC1 bacteria on the establishment of Leishmania major parasites in sandflies. P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of TC1 bacteria (1 × 10 in 5% sucrose solution). 8 CFU / ml) for 24 hours, while a control group of sandflies was fed 5% sucrose alone.

[0109] Sandflies were maintained on a diet of 30% sucrose ad libitum for 6 days, and then after an overnight starvation period (7 days after bacterial feeding), sandflies were inoculated with L. major promastigotes (5 × 10 6 Chicks were infected by artificial feeding through the membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml.

[0110] After infection, blood-fed females were selected and maintained on a 30% sucrose diet. At 7 and 11 days post-infection, sandflies were collected and the infection status was determined as previously described.

[0111] The data shown in Figure 5A indicate that exposure of sandflies to Delftia tsuruhatensis TC1 affects the establishment of Leishmania parasites in the sandfly midgut, even when exposure to the bacteria occurs 1 week before infection.

[0112] Overnight-starved P. duboscqi sandflies, 5–7 days old, were inoculated with L. major promastigotes (5 × 10 6 Sandflies were infected by artificial feeding through the chick membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 TC1 bacteria / ml. After infection, blood-fed females were selected and placed on a 30% sucrose diet. Five days after infection, when all blood residues had been excreted, sandflies were inoculated with a suspension of TC1 bacteria (1 × 10 in 5% sucrose solution). 8 The sandflies were fed cotton rolls soaked in 5% sucrose (CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. Sandflies were then maintained on a 30% sucrose diet ad libitum until the end of the experiment. At 7 and 11 days post-infection, sandflies were collected and the infection status was determined as previously described.

[0113] The data shown in Figure 5B indicate that exposure of pre-infected sandflies to Delftia tsuruhatensis TC1 affects the establishment of Leishmania parasites.

[0114] Overnight-starved P. duboscqi sandflies, 5–7 days old, were inoculated with L. major promastigotes (5 × 10 6 The chicks were infected by artificial feeding through the chick membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 TC1 bacteria / ml. After infection, blood-fed females were selected and placed on a 30% sucrose diet. Eight days after infection, when the infection had begun to mature, sandflies were inoculated with a suspension of TC1 bacteria (1 × 10 in a 5% sucrose solution). 8 The sandflies were fed cotton rolls soaked in 5% sucrose (CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. Sandflies were then maintained on a 30% sucrose diet ad libitum until the end of the experiment. Twelve days after infection, sandflies were collected and the infection status was determined as previously described.

[0115] The data shown in Figure 5C indicate that exposure of sandflies harboring mature infections to Delftia tsuruhatensis TC1 affects the establishment of Leishmania parasites.

[0116] Figure 6: Effect of different blood meals on Leishmania major parasites in sandflies fed Delftia tsuruhatensis TC1 bacteria. P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of TC1 bacteria (1 × 10 in 5% sucrose solution). 8 The sandflies were fed cotton rolls soaked in 5% sucrose (5 × 10 CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. After an overnight starvation period, the sandflies were inoculated with L. major promastigotes (5 × 10 6 Chicks were infected by artificial feeding through the membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml.

[0117] After infection, blood-fed females were selected and maintained on a 30% sucrose diet. 12 days after infection, sandflies were given another blood meal. Briefly, sandflies were blood-fed in anesthetized naive mice for 1 hour, and blood-fed females were selected and maintained on a 30% sucrose diet until the end of the experiment. After 6 days of another non-infected blood meal, sandflies were collected and infection status was determined as previously described.

[0118] The data shown in Figure 6 show that the infection rate in TC1-colonized sandflies surviving after another blood meal remains significantly lower than untreated controls, indicating that another blood meal does not reverse the infection phenotype.

[0119] Figure 7: Effect of bite infection of sandflies fed Delftia tsuruhatensis TC1 bacteria on infected skin lesions in mice with Leishmania major parasites. BALB / c mice were infected intradermally in the ear pinna with 1,000 L. major metastatic parasites and maintained with free access to water and food until the development of typical cutaneous leishmaniasis lesions. Sand flies were then blood-fed through the ear with active lesions using a vial with a mesh surface held in place by a custom-made clamp. The blood-fed sand flies were divided into two groups. One group was then fed a sugar meal (1 × 10 in 5% sterile sucrose solution) and the other was fed a sugar meal (1 × 10 in 5% sterile sucrose solution). 8 One group was fed with bacteria overnight at 5% sterile sucrose solution (1000 mg / ml), while the other received 5% sterile sucrose solution alone. Sandflies from both groups were dissected 11 days after infection and their infection status was assessed as previously described.

[0120] The data shown in Figure 7 indicate that when naturally infected by biting on infected skin lesions in mice, TC1-colonized sandflies exhibit reduced midgut parasite burdens and lower infection prevalence.

[0121] Figure 8: Sandfly mortality upon exposure to Delftia tsuruhatensis TC1 bacteria P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of TC1 bacteria (1 × 10 in 5% sucrose solution). 8CFU / ml) for 24 hours, while a control group of sandflies was fed 5% sucrose alone.

[0122] They were either not blood-fed, artificially fed with uninfected blood, or infected with L. major promastigotes (5 × 10 6 Bacteria-fed or control sandflies artificially fed with blood containing 100 flies / ml were placed in cardboard pints (100 flies per pint) and reared on a diet of 30% sucrose for 11–18 days.

[0123] Sandfly mortality was recorded daily, and dead sandflies were also removed daily. Furthermore, on day 12 postinfection, only infected sandflies were given another blood meal (as described above), and mortality was also recorded daily. Only selected blood-fed sandflies were considered for the next 6 days. Data are presented in the form of Kaplan-Meier curves and as dot plots for individual time points, with each symbol representing a group of 100 sandflies (for one blood meal) or a fraction of the total (for different blood meals). Statistical differences were determined using the nonparametric Kruskal-Wallis test with post-hoc analysis; values ​​of at least P ≤ .05 were considered statistically relevant.

[0124] The data are shown in Figure 8A-C. Exposure to TC1 bacteria slightly increases sandfly mortality but significantly affects the survival of infected sandflies. Mortality is significantly increased after another blood meal only in TC1-colonized sandflies infected with L. major parasites.

[0125] The data show that TC1 bacteria not only reduce the infectious load in sandflies, but also reduce the overall number of infected sandflies.

[0126] Figure 9: Experiments to establish whether the reduction in Leishmania major parasite load in sandflies is specific to live Delftia TC1 (a gram-negative bacterium) or whether it is applicable to all gram-negative bacteria. P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of Gram-negative, ampicillin-resistant E. coli or Ornithinibacillus massiliensis strains (1 × 10 in 5% sucrose solution) isolated from the microbiota of healthy P. duboscqi sandflies from our colony. 8 The sandflies were fed cotton rolls soaked in 5% sucrose (5 × 10 CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. After an overnight starvation period, the sandflies were inoculated with L. major promastigotes (5 × 10 6 Chicks were infected by artificial feeding through a membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml of HIV-1. After infection, blood-fed females were selected and placed on a 30% sucrose diet.

[0127] Sandflies were collected 7 and 11 days after infection (7 and 11 days, if applicable) and infection status was determined as previously described. The data shown in Figure 9A indicate that E. coli and Ornithinibacillus massiliensis do not have the same effect on parasite infection in the sandfly midgut as Delftia tsuruhatensis. The effect seen with TC1 is specific to TC1, not universal to Gram-negative bacterial infections.

[0128] Live TC1 bacteria were heat-inactivated at 95°C for 10 min to generate dead TC1 bacteria. P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of live or dead TC1 bacteria (1 × 10 in 5% sucrose solution). 8 The sandflies were fed cotton rolls soaked in 5% sucrose (5 × 10 CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. After an overnight starvation period, the sandflies were inoculated with L. major promastigotes (5 × 10 6Chicks were infected by artificial feeding through a membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml of HIV-1. After infection, blood-fed females were selected and placed on a 30% sucrose diet.

[0129] Eleven days after infection, sandflies were collected and the infection status was determined as previously described. The data shown in Figure 9B indicate that dead Delftia tsuruhatensis does not have the same effect as live Delftia tsuruhatensis on parasite infection in the sandfly midgut.

[0130] Figure 10 demonstrate the effect of Delftia tsuruhatensis in blocking the transmission of leishmaniasis to naive mice. P. duboscqi sandflies, 5–7 days old, were inoculated with a suspension of TC1 bacteria (1 × 10 in 5% sucrose solution). 8 The sandflies were fed cotton rolls soaked in 5% sucrose (5 × 10 CFU / ml) for 24 hours, while the control sandflies were fed 5% sucrose alone. After an overnight starvation period, the sandflies were inoculated with L. major promastigotes (5 × 10 6 Chicks were infected by artificial feeding through the membrane with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 1000 ng / ml.

[0131] After infection, blood-feeding females were selected and maintained on a 30% sucrose diet ad libitum. Eleven days after infection, 20 sandflies were placed on each ear of a BALB / c mouse using a mesh-surfaced vial held in place by a custom-made clamp and allowed to feed for at least 30 minutes. The number of blood-feeding flies was determined by observing them under a stereomicroscope. Mice were then monitored weekly to track the development of lesions caused by L. major infection.

[0132] Images of individual ears were acquired weekly using a smartphone (Figure 10A). Dey R, Joshi AB, Oliveira F et al., Gut microbes erupted during bites of infected sand flies augment the 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. Animals were euthanized 4 weeks after infection for determination of parasite burden by limiting dilution. Data are shown in Figure 10B. Data are presented in the form of dot plots, with each symbol corresponding to a single mouse ear. Statistical differences were determined using the nonparametric Mann-Whitney test, and a value of at least P ≤ 0.05 indicated statistical relevance.

[0133] Figure 10A: Mice bitten by leishmania-infected sandflies that had been pre-exposed to Delftia tsuruhatensis showed no ear lesions caused by L. major infection compared to mice bitten by infected sandflies that had not been exposed to Delftia tsuruhatensis (image from TEST panel).

[0134] Figure 10B: Parasite burden in the ears of mice bitten by leishmania-infected sandflies (bacteria) that had been pre-exposed to Delftia tsuruhatensis was significantly reduced compared to mice bitten by infected sandflies (controls) that had not been exposed to Delftia tsuruhatensis.

[0135] FIG. 10C: Sandflies "colonized" with TC1 bacteria were able to take more of another blood meal because the reduction in infection caused by TC1 led to a reduction in intestinal obstruction.

[0136] The data show that Delftia TC1 bacteria affect Leishmania major parasite development in P. duboscqi sandflies, as evidenced by significantly lower numbers (90% reduction) of both total parasites and infectious metaphase forms in bacteria-fed sandflies compared with control sandflies. This phenotype was consistently observed regardless of the timing of bacteria-feeding (1 week before infection vs. 1 day before infection vs. 5 days after infection) and was even enhanced in the context of sandflies fed a separate, non-infected blood meal. Furthermore, TC1 bacteria reduced not only the infectious load in sandflies but also the overall number of infected sandflies, resulting in increased mortality in Leishmania-infected sandflies but not in sugar-fed or blood-fed (non-infected blood meal) sandflies. The data also show that in the context of a mouse model of cutaneous leishmaniasis, Leishmania -infected TC1 bacteria-fed sandflies are less able to transmit Leishmania major parasites and cause disease (active lesions were observed in 25% of animals bitten by bacteria-fed flies compared to 80% in the control group, and parasites were detected in 27% of animals bitten by bacteria-fed flies compared to 100% of control animals).

[0137] Figure 11: Efficacy of harman in interfering with Zika virus replication in Aedes aegypti mosquitoes Approximately 10 mosquitoes carrying 3- to 5-day-old Aedes aegypti strain SBE were infected. 7 They were fed a blood meal (rabbit blood) containing PFU / ml of Zika virus and the indicated concentrations of harman dissolved in dimethyl sulfoxide (DMSO) via membrane feeders. The blood meal fed to the control group did not contain harman. Fully fed mosquitoes were selected and placed on a 10% sucrose diet. A total of 30 mosquitoes were analyzed per treatment.

[0138] After 7 days, the abdomen of the mosquito was dissected and placed in a 2 mL tube containing glass beads and 300 μL of DMEM medium. The sample was homogenized. Serial 1:10 dilutions of this solution were distributed into a 96-well plate containing VERO cells. After 5–6 days, the medium was discarded, and viral plaques were fixed and visualized with a staining reagent (1% crystal violet in 1:1 methanol / acetone solution) at room temperature for 1 hour.

[0139] Plates were rinsed with distilled water and air-dried. 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 represents the median. A dose-dependent decrease in PFU / mL was observed in the Harman-treated samples compared to the control group. Statistical differences were determined using the nonparametric Mann-Whitney test. No significant differences were observed in the treatment groups compared to the DMSO control.

[0140] The data in Figure 11 show reduced levels of Zika virus in mosquitoes that received harman.

Claims

1. 1. A composition for use in a method for reducing or preventing the transmission of a vector-borne disease, said composition comprising a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, said method comprising contacting at least one vector with said composition; The composition, wherein the vector-borne disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii disease, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, Ehrlichia muris-like disease, ehrlichiosis, filariasis, Heartland virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

2. 1. A composition for use in a method for reducing or preventing parasitic transmission, said composition comprising a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, said method comprising contacting at least one vector with said composition; The composition, wherein the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Borrelia mayonii disease, Chagas disease, Cryptosporidium, dirofilariasis, Ehrlichia muris-like disease, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific tick fever, Rocky Mountain spotted fever, and tularemia.

3. 3. The composition for use according to claim 1 or 2, wherein the bacterium is Delftia tsuruhatensis, and / or 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. 6. The composition for use according to any one of claims 1 to 5, wherein the composition is for use in reducing or preventing (i) Zika virus and / or (ii) Zika virus transmission.

7. 7. The composition for use according to claim 6, wherein the composition is for use in reducing or preventing the transmission of (i) Zika virus and / or (ii) Zika virus in mosquitoes.

8. 6. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is for use in reducing or preventing (i) Chikungunya virus and / or (ii) Chikungunya virus transmission.

9. 9. The composition for use according to claim 8, wherein the composition is for use in reducing or preventing the transmission of (i) Chikungunya virus and / or (ii) Chikungunya virus in mosquitoes.

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

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

12. 6. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is for use in reducing or preventing (i) Mayaro virus and / or (ii) Mayaro virus transmission.

13. 13. The composition for use according to claim 12, wherein the composition is for use in reducing or preventing the transmission of (i) Mayaro virus and / or (ii) Mayaro virus in mosquitoes.

14. 6. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is for use in reducing or preventing (i) West Nile Virus and / or (ii) West Nile Virus transmission.

15. 15. The composition for use according to claim 14, wherein the composition is for use in reducing or preventing the transmission of (i) West Nile Virus and / or (ii) West Nile Virus in mosquitoes.

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

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

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

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

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

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

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

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

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

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

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

27. 6. A composition for use according to any one of claims 2, 4 and 5, wherein the composition is for use in reducing or preventing (i) Cryptosporidium and / or (ii) the transmission of Cryptosporidium.

28. 1. A method for reducing or preventing the transmission of a vector-borne 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 method, wherein the vector-borne or parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii 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 tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

29. 1. 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 said vector-borne or parasitic disease, comprising: The vector-borne or parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, bourbon virus, Borrelia mayonii 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 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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