Anti-tick vaccine compositions and related methods

Abstract

Implementations of an anti-tick vaccine composition may include one or more conserved tick proteins from a tick species; and one or more conserved outer membrane proteins of an endosymbiont of the tick species.

Description

ANTI-TICK VACCINE COMPOSITIONS AND RELATED METHODSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This document claims the benefit of the filing date of U. S. Provisional Patent Application 63 / 714,050, entitled “Anti-Tick Vaccine Compositions to Prevent Tick-Related Diseases” to Wagner et al. which was filed on 10 / 30 / 2024, the disclosure of which is hereby incorporated entirely herein by reference.SEQUENCE LISTING

[0002] This document contains the material in and hereby incorporates entirely herein by reference the sequence listing file in XML format filed herewith named NAU115.xml, created 10 / 30 / 2025, which is 328,316 bytes in size.BACKGROUND1. Technical Field

[0003] Aspects of this document relate generally to vaccines such as compositions and methods for preventing animals from becoming hosts to ticks and reducing overall tick populations.2. Background

[0004] Ticks and tick-borne diseases are a significant and increasing global problem for human and animal health. Human diseases like Rocky Mountain Spoted Fever and Lyme disease are transmitted via ticks. Animal diseases like babesiosis, bovine anaplasmosis, and cattle fever are transmitted through ticks as well.SUMMARY

[0005] Implementations of an anti-tick vaccine composition may include one or more conserved tick proteins from a tick species: and one or more conserved outer membrane proteins of an endosymbiont of the tick species.

[0006] Implementations of an anti-tick vaccine may include one, all, or any of the following:

[0007] The one or more conserved tick proteins and the one or more conserved outer membrane proteins each have a conservation level equal to or greater than 97%.

[0008] The tick species may be Rhipicephalus microplus and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 1-28 and any combination or fragment thereof and the endosymbiont may be CLERM and the one or more conserved outer membrane proteins may be selected from the group consisting of SEQ ID NO: 190-207 and any combination or fragment thereof.

[0009] The tick species may be Rhipicephalus sanguineus sensu lato and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 29-56 and any combination or fragment thereof and the endosymbiont may be CLERS and the one or more conserved outer membrane proteins may be selected from the group consisting of SEQ ID NO: 221-232 and any combination or fragment thereof

[0010] The tick species may be Amblyomma americanum and the one or more conserved tick proteins maybe at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 113-140 and any combination or fragment thereof and the endosymbiont may be CLE AA and the one or more conservedouter membrane proteins may be selected from the group consisting of SEQ ID NO: 233-242 and any combination or fragment thereof.

[0011] The endosymbiont may be CLERA and the one or more conserved outer membrane proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 208-220 and any combination or fragment thereof.

[0012] The tick species may be Ixodes scapularis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%. or 100% identical to a protein selected from the group consisting of SEQ ID NO: 57-84 and any combination or fragment thereof.

[0013] The tick species may be Ixodes pacificus and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 85-112 and any combination or fragment thereof.

[0014] The tick species may be Haemaphysalis longicomis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 141-161 and any combination or fragment thereof.

[0015] The tick species may be Dermacentor variabilis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 162-189 and any combination or fragment thereof.

[0016] Implementations of a method of vaccinating an animal against hosting a tick may include administering to the animal a pharmaceutically effective amount of a bivalentvaccine including one or more conserved tick proteins from a tick species and one or more conserved outer membrane proteins of an endosymbiont of the tick species.

[0017] Implementations of a method of vaccinating an animal may include one, all, or any of the following:

[0018] Administering may include injecting or delivering the vaccine into a body or mucosa of the animal or oral application to the animal and where the bivalent vaccine further may include where the one or more conserved tick proteins and the one or more conserved outer membrane proteins each have a conservation level equal to or greater than 97%.

[0019] The tick species may be Rhipicephalus microplus and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 1-28 and any combination or fragment thereof and the endosymbiont may be CLERM and the one or more conserved outer membrane proteins may be selected from the group consisting of SEQ ID NO: 190-207 and any combination or fragment thereof,

[0020] The tick species may be Rhipicephalus sanguineus sensu lato and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 29-56 and any combination or fragment thereof and the endosymbiont may be CLERS and the one or more conserved outer membrane proteins may be selected from the group consisting of SEQ ID NO: 221-232 and any combination or fragment thereof.

[0021] The tick species may be Amblyomma americanum and the one or more conserved tick proteins may be at l east 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 113-140 and any combination or fragment thereof and the endosymbiont may be CLEAA and the one or more conservedouter membrane proteins may be selected from the group consisting of SEQ ID NO: 233-242 and any combination or fragment thereof.

[0022] The endosymbiont may be CLERA and the one or more conserved outer membrane proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 208-220 and any combination or fragment thereof.

[0023] The tick species may be Ixodes scapularis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%. or 100% identical to a protein selected from the group consisting of SEQ ID NO: 57-84 and any combination or fragment thereof.

[0024] The tick species may be Ixodes pacificus and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 85-112 and any combination or fragment thereof.

[0025] The tick species may be Haemaphysalis longicomis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 141-161 and any combination or fragment thereof.

[0026] The tick species may be Dermacentor variabilis and the one or more conserved tick proteins may be at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 162-189 and any combination or fragment thereof.

[0027] The foregoing and other aspects, features, and advantages will be apparent to those artisans of ordinary skill in the art from the DESCRIPTION and DRAWINGS, and from the CLAIMS.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Implementations will hereinafter be described in conjunction with the appended drawings, where like designations denote like elements, and:

[0029] FIG. 1 is an amino acid distance metric for various proteins from a study of diversity of Rhipicephalus microplus ticks from a variety of geographic locations;

[0030] FIG. 2 is a chart of the amino acid similarity percentage among a set from proteins from R. microplus and Rhipicephalus. Sanguineus ticks;

[0031] FIG. 3 is a diagram of a selection and down -selection process for a set of Ixodes scapular is tick proteins and for the corresponding endosymbiont Rickettsia buchneri proteins;

[0032] FIG. 4 is a diagram of an implementation of a process of utilizing an animal model to assess the vaccine potentials for tick proteins and endosymbiont proteins;

[0033] FIG. 5 is a diagram of an implementation of a process of utilizing a rabbit, mouse, and white-tailed deer models to evaluate tick proteins and endosymbiont proteins to form bivalent vaccine candidates;

[0034] FIG. 6 is a timeline of various experimental activities in a process for evaluating a protein in a rabbit model;

[0035] FIG. 7 is a timeline of various experimental activities in a process for evaluating a protein in a white-tailed deer model;

[0036] FIG. 8 is a timeline of various experimental activities in a process for evaluating a protein in a mouse model;

[0037] FIG. 9 is a diagram of a first portion of a process for evaluating CLERM proteins for testing for use in a vaccine composition;

[0038] FIG. 10 is a diagram of a second portion of a process for evaluating CLERM proteins for testing for use in a vaccine composition;

[0039] FIG. 11 is a diagram of a process of selecting CLERM proteins for evaluation in a cattle study;

[0040] FIG. 12 is a timeline of various experimental activities in a process for evaluating a protein in a cattle model;

[0041] FIG. 13 is a graph showing locations of field collections of R. microplus in North and Central America; and

[0042] FIG. 14 is two diagrams of the overlap genes in the genomes of three CLERS genomes and a CLERM genome, CLERS genome, and Coxiella burnetii genome.DESCRIPTION

[0043] 1'his disclosure, its aspects and implementations, are not limited to the specific components, assembly procedures or method elements disclosed herein. Many additional components, assembly procedures and / or method elements loiown in the art consistent with the intended anti-tick vaccine compositions and related methods will become apparent for use with particular implementations from this disclosure.Accordingly, for example, although particular implementations are disclosed, such implementations and implementing components may comprise any shape, size, style, type, model, version, measurement, concentration, material, quantity, method element, step, and / or the like as is known in the art for such anti-tick vaccine compositions and related methods, and implementing components and methods, consistent with the intended operation and methods.

[0044] Anti-tick vaccines target tick proteins. Anti -tick vaccines represent an alternative to acaricide-based tick control. The strategy of an anti-tick vaccine is to immunize a host (dog, cad, cattle, deer, Or native mice) with tick antigens that stimulate a strong humoral IgG antibody response. Antibodies from the vaccinated animal then enter a tick via its blood meal and bind to corresponding protein targets in the gut, salivary gland,or ovary, negatively impacting the tick’s physiology and sometimes causing mortality. An important outcome is to weaken ticks and reduce their reproductive output, so that successive tick generations are reduced to the point that disease transmission breaks down or becomes minimal. In selecting the proteins, prioritizing “concealed” antigens within ticks that a host immune system normally would not encounter is an important consideration because these protein(s) occur inside the tick body (midgut, hemolymph, ovary, etc.).[00451 IgG antibodies in host blood retain their reactivity outside of the host (inside a tick) and, although not usually bactericidal by themselves, can also neutralize endosymbiont bacteria by binding and inhibiting abundant cell surface proteins on the bacteria. For example, host antibodies induced acutely negative effects and even mortality in Ixodes ricinus ticks fed on transgenic mice vaccinated with E. coli, IgG antibodies can cross the tick midgut and enter the hemolymph where they can bind to vaccine targets and persist through metamorphosis to nymphal or adult stages. IgG antibodies can even reach tissues such as salivary glands, ovaries and eggs, and muscle,

[0046] Anti-microbiota vaccines target bacterial species (some of which are endosymbionts) that live inside of ticks. A complementary strategy for anti-tick vaccines is to target tick microbiota that provision ticks with required nutrients. This approach can weaken or even kill the ticks and thereby disrupt pathogen transmission. Obligate members of the tick microbiome include bacterial endosymbionts that are maternally inherited. These maternally inherited bacterial endosymbionts are often the most dominant species wi thin a tick and have great potential as targets of pest control in their own right.Endosymbionts exist because ticks feed solely on blood, an incomplete source of certain nutrients, especially B vitamins and amino acids. As such, these essential nutrients must be supplied by one or more endosymbiont genera present in the gut microbiome, includingCoxiella, Francisella, and Ricketsia species. The paper by Duron et al. entitled “Evolutionary changes in symbiont community structure in ticks,” Molecular Ecology’ V.26, p. 2904-2921 (2017, doi: 10.111 l / mec.14094), the disclosure of which is hereby incorporated entirely by reference, discusses many of the characteristics and distributions of various endosymbionts in various tick species. GoxieZZn-like endosymbionts (CLEs) are important in many genera of ticks including Rhipicephalus. Indeed, the CLE in R. microplus (referred to herein as “CLERM”) is required for development past the metanymph stage, and experimental elimination of the CLERM has demonstrated downregulation of multiple genes that are important for blood feeding and likely other factors. Experimental reduction of the CLE of the highly invasive Asian longhorned tick Haemaphysalis longicomis using tetracycline led to a decline in tick feeding behavior due to decreased serotonin, as the precursor for tick serotonin is provided via a shikimate pathway for chorismate in its CLE as described in the paper to Zhong et al, “Symbiont-regulated serotonin biosynthesis modulates tick feeding activity,” Cell Host &. Microbe, V.29, p. 1545-1557 (October 13, 2021, doi.org, '10.1016 / j.chom.2021.08.011). Other tick species may also rely on precursors provided using a shikimate pathway including those that utilize Francisella species as endosymbionts as the existence of such a pathway is disclosed in the paper to Wagner et al., “Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples,” PLOS One, p. 1-30 (October 12, 2022, doi.org / 10.1371 / journal.pone.0273273), the disclosure of which is hereby incorporated entirely herein by reference.

[0047] The obligate bacterial endosymbionts targeted in the vaccine implementations disclosed herein are generally intracellular and live inside arthropod cells of the Malpighian tubules, midgut, and ovaries. There is a high probability that antibodies targeting endosymbiont outer membrane proteins (OMPs) can enter tick cells and attackthese bacteria. Multiple studies have demonstrated that antibodies against intracellular tick targets must pass through the cell membrane to be effective and this was experimentally confirmed using ISE6 tick cell culture. In fact, of seven published vaccine candidates focused solely on intracellular tick proteins, all produced significantly negative effects in ticks that fed on vaccinated hosts and the greatest efficacy reported (90%) was conveyed by the ribosomal PO antigen. The other reported intracellular candidates include subolesin, VDAC, ferretin-1, Hc-23, interphase cytoplasm foci protein (2G7), and troponin P27 / 30.

[0048] Mechanisms of antibody access to tick cells include 1 ) direct uptake into midgut epithelial cells via phagocytosis during blood meal processing, or 2) circulation in the tick hemolymph and distribution to other tissues where antibodies can subsequently enter cells. Another mechanism that further allows additional access of IgG antibodies to intracellular endosymbionts is that vaccines such as Bm86 and other midgut antigens are known for their ability to raise a strong IgG response that can severely damage tick midgut tissue, possibly by activating the complement cascade of the innate immune response. The resulting damaged or lysed cells should release endosymbiont bacteria, thus also exposing them to circulating IgG antibodies. Finally, endosymbionts spend part of their time outside of host cells and retain the ability to re-enter host cells, which suggests they are not intracellular at all times. They can circulate in hemolymph when migrating to ovarian tissues, as observed in the Rickettsia endosymbiont of whiteflies. This translocation is critical for ensuring vertical transmission to eggs and embryos. During periods outside of host cells, endosymbionts would be vulnerable to binding by host IgG antibodies received by the tick from a bloodmeal. A dual-action or bivalent vaccine that uses one or more tick proteins in combination with and one or more endosymbiont proteins may provide maximum access for endosymbiont and tick vaccine targets inside cells. By “in combination"’ is meant a vaccine where the composition includes both one or more tickproteins and one or more endosymbiont proteins which are administered at the same time. It also means a vaccine where the one or more tick proteins is included in a composition that is administered first followed by administration of a composition that contains the one or more end osymbiont protein and vice versa. The goal of the combination vaccine i s that the IgG antibody effects of the vaccine will be felt by the host at the same time so as to enable ticks feeding from a host to acquire IgG antibodies for both the tick protein(s) and the endosymbiont protein(s) in their next single blood meal. In this document, the focus is on outer membrane proteins (OMPs) of endosymbionts as vaccine targets, because they will be most accessible to any host IgG antibodies that are able to enter tick cells or come into contact with endo symbionts outside of tick cells. However, it is possible in other vaccine implementations that other proteins internal to the endosymbionts may also be selected.[0049 An additional consideration for anti-tick vaccines is to utilizes highly conserved protein targets for both the tick protem(s) and endosymbiont protein(s) selected. The first anti- tick vaccines for catle used tire Rhipicepkahis microplus tick protein Bm86 and were implemented in the early 1990s. Vaccines marketed under the tradenames TICKGARD® by Pastoral AG of Adelaide, South Australia and GA VAC® by the Center for Genetic Engineering and Biotechnology of Camaguey, Cuba initially provided 55-100% control of R. microplus and Rhipicephalus annulatus (depending on the tick population), thereby reducing the number of acaricide treatments needed for a herd by 60% on average. However, the molecular basis of these first-generation vaccines was poorly understood and there are geographic differences in the efficacy of Bm86-based vaccines on different tick populations (resulting in 0- 1% tick control), likely due to antigenic variation within key epitopes of this locus. FIG. 1 illustrates how, using a distance metric from a study of diversity off?, microphis ticks from a variety of geographic locations the Bm86protein is one of the least conserved, but others like VDAC, AQP1, and V R are highly conserved. Indeed, accounting for protein variation is important to prevent “vaccine escape” by different groups of a species population a problem that achieved widespread public awareness during the SARS-CoV-2 vaccination campaign. The bivalent vaccine implementations disclosed herein target proteins that are highly conserved in both the tick and endosymbiont and, thus, useful against all populations of a single (or multiple) tick species. A bivalent vaccine can also be highly robust to immune escape, requiring mutations in both the tick and endosymbiont genomes for vaccine resistance to evolve, and therefore should have greater long-term efficacy against ticks.

[0050] The present implementations of vaccine compositions designed for use in methods of vaccinating an animal against hosting a tick utilize at least one protein from a t least one tick species at least one protein from at. least one endosymbioni of the at least one tick species. Specific protein implementations for the endosymbionts utilize outer membrane proteins. Both the at least one protein from the tick and the at least one protein from the endosymbiont are selected based on how highly conserved they are among different populations of the tick species, hi some implementations, the at least one protein from the tick and the at least one protein from the endosymbiont are highly conserved meaning greater than or equal to 97% amino acid similarity across many different tick populations. In some implementations, the tick protein targets may be selected based on an expectation that the protein targets will appear in 100% of ticks in the different tick populations. Referring to FIG. 2, a diagram showing the amino acid similarity between geographically dispersed tick populations of R. micropius and S. sanguineus shows a number of proteins above the 97% threshold and also indicates that the same protein is conserved across tick species to a similar degree (see VDAC). The lower the similarity percentage the lower the conservation of the protein which, as indicated in FIG. 2 alsomeans there is a higher risk of vaccine escape for these proteins. Bm86 is illustrated to have less than a 90% amino acid conservation in the full-length protein, meaning that a vaccine based on it might have reduced efficacy in different tick populations allowing a large number of remaining ticks to successfully infest vaccinated hosts and pass on their specific genetic signature.

[0051] Examples of highly conserved tick proteins include, by non-limiting example, VDAC, AQP1, VgR, Serpin 1, Sub and Ribosomal P0. In the ease of Vitellin, evidence that this gene is associated with Rickettsia endosymbionts has been observed. For Bm86, evidence that that the corresponding antibodies cause "physical damage to the tick midgut has been observed. Preliminary data has also indicated three locations on the Bm86 protein where an IgG antibody can bind to it, further increasing the odds that an IgG antibody will find a positive interaction with the protein in a tick.

[0052] Non-limiting examples of outer membrane proteins (OMPs) of Coxiella and Rickettsia endosymbionts that may be utilized in a vaccine include OnipA, OmpB, OmpH, EF-tu, and YbgF. The OmpA and OmpB proteins are abundant on the outer membrane of Gram-negative bacteria. The OmpH and EF-tu proteins are expressed in the adult female life stage of R. microplus ticks. The YbgF protein is a vaccine target against the pafhogenCo. / fo btirn&tiid, and also found in endosymbiont bacteria.

[0053] Examples of tick species for which the vaccine compositions disclosed herein may be directed follow. In some implementations, a single vaccine composition may be used to target multiple tick species or a single tick species depending upon the tick protein used and the specific endosymbiont being targeted as well. Many other tick species other than those listed here that target various animals may be targeted in vaccine compositions using the principles disclosed herein.

[9054] In particular implementations, the tick species may be Rlriplcephdlus sanguineus. This species is commonly known as the brown dog tick, but is actually a complex of multiple different species of ticks with multiple variants in the United States and globally. In this document, the combination of species and variants referred to as brown dog ticks will be referenced as Rhipicephalus sanguineus ensu lata (s. L) to include all of these variations. The obligate nutritional endosymbiont of R. sanguineus s. I. is a Coxiella-like bacteria referred to herein as the Coxiella-like endosymbiont of R.Sanguineus or CLERS. It is possible that an intact shikimate pathway exists with the CLERS organism. The hosts targeted with the vaccine implementation would be dogs and cats and likely would be administered as an injectable formulation or deliverable to mucosal surfaces through other mechanisms, such as a gene gun.

[0055] In particular implementations, the tick species may be Rhipicephalus microplus. This tick species is commonly referred to as a cattle fever tick. The obligate nutritional endosymbiont for 7?. microplus is the Coxiella-like endosymbiont of R. microplus or CLERM. It is possible that an intact shikimate pathway exists with the CLERM organism. In other implementations, the tick may be Rhipicephalus anniilatus or Rhipicephalus appendiculatus which are also commonly referred to as cattle fever ticks. The obligate nutritional endosymbiont for R. anniilaiUs is referred to herein as the Coxiella-like endosymbiont of R. annulaius or CLERAN. The obligate nutritional endosymbiont for R. appendiculatus is referred to as the Coxiella-like endosymbiont of R. appendiculatus or CLERAP. The hosts for the vaccine implementations directed toward these ticks would be cattle and would likely be administered as an injectable formulation or deliverable to mucosal surfaces through other mechanisms, such as a gene gun.

[9056] In particular implementations, the tick species may beIxodes scapularis. This tick species is commonly referred to as the black-legged tick or deer tick and is theprimary vector of Lyme disease along with other pathogens in the United States. The obligate nutritional endosymbiont of I. scapularis is Rickettsia buchneri. No intact shikimate pathway is currently known for this endosymbiont. The target hosts for this tick species are white-tailed deer and small mammals including mice and rabbits. Because the target hosts are wildlife, edible oral baits are the method of administration of the vaccine composition for this tick species.

[0057] In particular implementations, the tick species may be Haemaphysalis longicorttis. This tick species is commonly known as the Asian loflghomed tick which has only recently been introduced into the United States but is spreading rapidly. While this tick species is known to transmit pathogens the primary issue experienced by hosts is that the number of ticks present on the host can become extremely high leading to weight loss, anemia, and reduced production in the case of various livestock. The obligate nutritional endosymbiont of. longicornis is a Coxiella-Like endosymbiont of. longicornis or CLEHL. An intact shikimate pathway exists with the CLEHL organism. While cattle are the primary host target for the vaccine, there are other animals who may benefit from treatment. Administration of a vaccine directed toward this tick species would be primarily an injectable formulation or deliverable to mucosal surfaces through other mechanisms, such as a gene gun.

[0058] In particular implementations, the tick species may be Ambfyomma mericanum. This tick species is commonly known as the lone star tick. While this tick species can transmit diseases, the main health risk to humans of this tick is that it is the primary source of alpha-gal syndrome in humans. Alpha-gal syndrome is induced in a human host following a bite from a lone star tick because galactose-a~l,3~galactose or alpha-gal is produced in the saliva of the tick. The human body then produces antibodies to alpha-gal. Since alpha-gal is also found in red meat products, this means that the host’santibodies can cause a serious and potentially life-threatening allergy to the red meat products going forward (which is why the allergy is also called the red meat allergy or tick bite meat allergy). As the vaccine compositions disclosed herein are not directed specifically for human use, the ability to target the animals who host the lifecycle of A. americanum will be key. The obligate nutritional endosymbiont of A. americanum is a Coxiella-like endosymbiont of A. Americanum or CLEAA. It is possible that an intact shikimate pathway exists in the CLEAA organism. The administration method for the vaccine preparations is oral through baits distributed so wildlife can consume them.[0059} In particular implementations, the tick species may be either Dermacen lor variabilis or D. similis. These ticks are commonly known as the American dog tick.Historically the D. similis population on the west coast of the United States was regarded as a distinct population of D. variabilis but was recently reclassified as a separate species. However, past and current publications lump the two species together. The obligate nutritional endosyiiibiont for both tick species is a Francisella-like endosymbiont ofD. variabilis or FLEDV. The host targets for the vaccine would be dogs and cats so the vaccine composition would be administered using an injectable formulation or deliverable to mucosal surfaces through other mechanisms, such as a gene gun.

[9060] In particular implementations, the tick species may be Ixodes pacificus. This tick is commonly known as the western black-legged tick and is the primary vector of Lyme disease on the west coast of the United States. The obligate nutritional endosymbiont for I. pacificus is a Rickettsia endosymbiont of I pacificus or REIP. This endosymbiont is also known as Rickettsia moriace sis str. Humboldt according to the paper by Alowaysi et at entitled “Isolation and Characterization of a Rickettsia from the Ovary of a Western Black-legged Tick, Ixodes pacificus ” Ticks Tick Borne Dis. V. 10, No. 4, p.918-923 (June 2019, dot: 10.1016 / j.ttbdis.2O 19.04.01.7) the disclosure of which is herebyincorporated entirely herein by reference. REIP is also known as Ricketsia species phylotype G021 according to the paper by Hunter et al., “The Ricketsia Endosymbiont of Ixodes paciftctts Contains All the Genes of De Novo Folate Biosynthesis,” PLOS ONE V.10, No. 12, p. 1-15 (12 / 9 / 2015, DOI:10.1371 / ournai.pone.0i44552, the disclosure of which is hereby incorporated entirely herein by reference. It is not believed that an intact shikimate pathway exists for this organism. A number of different hosts are used by this tick and so vaccination compositions may be administered orally via baits or perhaps via injections depending on the host animal targeted or deliverable to mucosal surfaces through other mechanisms, such as a gene gun.[0061 J V 'arious compositions of vaccines that contain one or more tick proteins and one or more corresponding endosymbiont proteins can be constructed using the principles disclosed in this document. The various compositions may include one, all, or any of the following tick proteins or fragments thereof from Rhipicephalus microplus listed in Table 1 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Deuteh F79, genome WOVZOOOOOOOO. I, with a mitochondrial genome of KP 143546.Table 1

[0062] The various compositions may include one, all, or any of the following tick proteins of fragments thereof from Rhipicephahis sanguineus sensu lato listed in Table 2 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Rsan-2018, genome JABSTVOl 0000000, with a mitochondrial genome of OQ 184024.Table 210063 j The various compositions may include one, all, or any of the following tick proteins or fragments thereof from Ixodes scaptddris listed in Table 3 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain PalLabHiFi, genome ABJBO 10000000, with a mitochondrial genome of MZ645749.Table 30064] The various compositions may include one, all, or any of the following tick proteins or fragments thereof from Ixodes pacificus listed in Table 4 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain CFI_29930, genome CAXMZBOOOOOOOOO, with a mitochondrial genome of PQ557592.Table 4

[0065] The various compositions may include one, all, or any of the following tick proteins or fragments thereof fxoi Ambfyomma americanum listed in Table 5 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain F_SG_1, genome JBQHYB 000000000,1, with a mitochondrial genome of NC 027609.Table 5

[0066] The various compositions may include one, all, or any of the following tick proteins or fragments thereof from Haemaphysalis longicornis listed in Table 6 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain HaeL-2018, genome JABSTR000000000.1, with a mitochondrial genome of PQ380096. ETable 6

[9067] The various compositions may include one, all, or any of the following tick proteins or fragments thereof from Dermacentor variabilis listed in Table 7 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Ectoservices, genome JBNOKL. OOOOOOOOO, with a mitochondrial genome of NC_061217.Table 7

[0068] The various compositions may include one, all, or any of the following endosymbiont proteins or fragments thereof from CLERM listed in Table 8 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Deutch F79, genome DLUJ00000000.Table 8[0069 J The various compositions may include one, all, or any of the following endosymbiont proteins or fragments thereof from CLERA listed in Table 9 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Klein Grass.Table 9

[0070] The various compositions may include one, all, or any of the following endosymbiont proteins or fragments thereof from CLERS listed in Table 10 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain Rsan-2018 with genome NIQBOOOOOOOO.Table 10

[0071] The various compositions may include one, all, or any of the following endosymbiont proteins or fragments thereof from CLEAA listed in Table 11 below with their corresponding SEQ ID NOs. These tick proteins are taken from strain F_SG_1 with genome NZ_CP007541.1Table 11

[0072] While the sequences in the foregoing tables have included the whole proteins, in various implementations, specific peptide sequences or fragments from the whole proteins may be what are ultimately used in various vaccine compositions. As used herein, the term “fragment” refers to a portion of a protein substantially identical to any of the reference proteins disclosed herein (whether through sequence listing or otherwise) that retains the biological acti vity7of the reference protein. In various implementations, the fragment may retain at least 50%, 75%, 80%, 90%, 95%, or 99% of the biological activity of the reference protein. Thus, this document contemplates that proteins from ticks and endosymbionts may be selected from peptides that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequences disclosed herein or fragments thereof. Furthermore, in various implementations, the tick and / or endosymbiont proteins can be purified proteins or encoded by nucleic acids (DNA or RNA) introduced into the cells of the animal.

[0073] Various techniques and methods may be used to develop and determine the particular tick proteinfs) and endosymbiont protein(s) selected for use in a vaccine composition like those disclosed herein. A diagram of the selection and down-selection process for the tick I. seapularis and the corresponding endosymbiont R. buchneri is illustrated in FIG. 3. hi FIG. 3, the tick protein process is illustrated on the left and involves examining new and published anti -tick protein targets to come up with an initial list of proteins (here over 60 potential targets). The list of protein targets is then analyzedusing various techniques disclosed later herein including animal testing to identify those targets that have greater than 70% efficacy in affecting a tick using various metrics including death, inability to reproduce, etc. This reduced list is then analyzed to pick those tick proteins that are identified as being expressed in multiple tick tissues, which significantly increases the odds that IgG antibodies from a blood meal will affect the tick. In various method implementations, an analysis of predicted B-cell epitopes at cell surfaces is also carried out to help refine the list of proteins to those who can attack the cell membrane structures directly and avoid selecting proteins that are mainly effective once they have entered a tick cell. Referring to the down-selection process on the right, a similar process is used to identify proteins for the endosymbiont j?, btichneri with the difference that since less is known about what proteins from this organism will generate an IgG antibody in the first place, the process begins with whole cell immunization into a model animal followed by identifying the antigens produced. The process then involves linking the identified antigens to outer membrane (OM) proteins with homology to the related organism R. ricketfsii (the organism that causes Rocky Mountain spotted fever in humans and is more well understood). Since outer membrane proteins are being targeted, the next step is to perform predictions of B~cell epitopes at. the cell surface to screen out any proteins that do not appear to target a protein on the cell surface. The resulting list of proteins is then compared to a list of other Rickettsial endosymbion ts in other tick species to evaluate the conservation of each protein to pick one with the highest likelihood of reaching a maximum number of ticks and tick species to reduce the odds of vaccine escape.

[0074] A screening of 30 adult female / , scapuldris ticks obtained from various locations in the United States for the presence of R. buchfterf indicated that this bacterium was present in all of the 30 ticks (100%). Thus, a focus on selecting proteins from R.buchneri for I. scapuiaris ticks would be a way to ensure conservation of the endosymbiont proteins.

[0075] Further detail with respect to the / buchneri process follows. The New Zealand white rabbit is a leading model for anti-tick vaccine studies. In a first trial, rabbits purchased from a commercial supplier are placed into one control group and two experimental groups (n=3 each) will be used to test two recombinant proteins (I scapularis Tnl and R. buchneri OmpB) using an adjuvant marketed under the tradename TITERMAX GOLD by TiterMax US A, Inc. of Norcross, Georgia. Intramuscular vaccinations with 50 micrograms of antigen and tick infestations will fallow the timeline in FIG, 6 including evaluating immunization with whole-cell R. buchneri, Live adult 7. scapularts can be obtained from the Oklahoma State University tick rearing facility. Serum from the rabbits can be drawn to quantify the IgG titer to each antigen, and 16S analysis can be performed using 10 partially fed females from each rabbit. Sera monitoring during the experiment ensures that vaccinated rabbits have raised a significantly higher level of IgG antibody compared to control animals. A sui table cutoff point in a particular imp lementation is an OD 450 nm reading of 0.35 for a 1 / 500 dilution of serum. With the adult ticks, the effects on the reproductive fitness of females based on weight, egg mass weight, and egg hatching success are then quantified including statistical comparisons between control vs. vaccinated groups. The foregoing process is summarized in FIG. 4.

[0076] If each antigen demonstrates effectiveness by itself (i.e., a significant reduction in the aforementioned measurements of female reproductive fitness for ticks feeding on vaccinated hosts compared to control group ticks), a second trial can be performed to vaccinate rabbits (n=3) using both vaccine protein targets (i.e., tick Tnl + endosymbiont OmpB) concurrently. The two antigens will be delivered at the same time onopposite sides of the body together with TITERMAX GOLD ad juvant. All other methods for trial #2 can be identical to the first trial.

[0077] This process can then be adapted to testing the resulting tick and endosymbiont paired proteins for effectiveness in additional animal models. As illustrated in FIG. 5, following identification of one or more pairs of proteins in the rabbit model using the preceding approach, a mouse model and then a deer model are challenged. The mouse model focuses specifically on observing the effect s of the vaccine on tick nymphs feeding on mice because the life cycle of I. 'Scapularis wvG f^ the ticks feeding on small mammals first and then moving to deer as they age and become adults.

[0078] A representative testing approach and schedule for the mice (. leucopus) is illustrated in FIG. 8. hi this testing approach, up to five I. scapuldris and five R. buchnen proteins can be tested in Peromyscus leucopus mice using the most protective proteins from IT2 and IT3. A small pilot study with one full-length protein can be used to determine an appropriate subcutaneous dose for mice ('20 micrograms of antigen) and verify immunogenicity using TiterMax Gold adjuvant. Mice can then be placed into 10 experimental and 4 control groups <n=3 / group; 42 total). Individual recombinant proteins are used to immunize each subject, with a booster at day 21 (FIG. 8); control animals will receive lx PBS in TITERMAX GOLD adjuvant. A small blood sample (100 microliters) can be collected at five time periods (FIG. 8) during the experiment to quantify antibody responses through time using enzyme-linked immunosorbent assay (ELISA). At the end of the experiment, the mice are euthanized and serum collected via a terminal blood draw. On day 31, each mouse can be challenged with 201, scapularis nymphs. Mice will be sedated briefly with isoflurane so ticks can be applied to the dorsal neck area; ticks will be attached for the next 4 hours while mice are kept in wire-mesh tubes wrapped with stockinete. Ticks will be allowed to feed until they engorge and / or drop from the host, about 4 days forthe nymphal stage. Detached ticks are collected daily and weighed. The effects of immunization on bloodmeal processing efficiency by measuring tick mortality, days to detachment, and weight upon detachment are then quantified for the various proteins being tested.

[0079] Following the testing in mice, testing of the best performing tick and etdosymbiont proteins can then be conducted using vaccinations of the tick proteins and endosymbiont proteins individually followed by testing using vaccinations of the tick proteins and endosymbiont proteins combined. Young deer white-tailed deer (WTD) can be placed into 3 experimental and 1 control groups (n=3 / group; 12 total ). Deer will be given intramuscular vaccinations (200-400 micrograms of protein) and tick infestations will follow the timeline in FIG. 7 using TH ERMAX GOLD adjuvant. A 10 L blood sample will be obtained at five time points during the experiment to quantity antibody responses through time using ELISA. Sera monitoring during the experiment is used to determine if vaccinated WTD raise a significantly higher level of IgG antibody than control animals; a suitable cutoff is an OD 450 ran of 0.35 for a 1 / 500 dilution ofserum. A second booster will be given if needed, which will extend the timeline by 3 weeks.

[0080] On day 31, each deer can be challenged with 1001, scapularis nymphs and 100 adults (50 females / 50 males). Ten of the adult female I. scapuldris from each WTD are collected after three days of feeding to analyze their microbiome community, leaving 40 females / 50 males for the reproduction study. Host animals will be prepared for infestation by shaving hair from a small dorsal area o f the thorax and gluing pouches to the skin. Ticks will be introduced via the resealable pouch opening and allowed to feed until they engorge and / or drop from the host, about 4 days for juvenile stages and about 6-10 days for adult females. Detached ticks are removed daily and weighed. Adult female ticks are incubated for a study of reproductive fitness as the same as previously described. DNA is thenextracted from pooled samples of newly hatched I. scapula ris larvae to perform PCR-based relative quantification of / ?, buchneri (CKP3) in larvae originating from females fed on immunized vs. control hosts. If individual antigens demonstrate strong effects (i.e., a significant reduction in the aforementioned measurements of female reproductive fitness for ticks feeding on vaccinated hosts compared to control group ticks), a second trial using vaccination of pairs of protein targets concurrently (combined vaccine) is conducted. Deer are placed into 3 experimental and 1 control groups (n=3 / group; 12 total). Intramuscular vaccinations with an appropriate amount of each antigen (200-400 micrograms) are delivered a t the same time on opposite sides of the body using TITERMAX GOLD adjuvant (half volume / site). Tick infestations and 10 mL blood draws are then conducted using the timeline in FIG. 7, Sera monitoring continues during the ex eriment to determine if vaccinated WTD raise a significantly higher level of IgG antibody than control animals and whether an additional booster is needed. The timeline of the trial of the combined vaccine is then extended to perform repeated tick infestations (3 total) on vaccinated and control deer, with each infestation separated by 1 month (not shown in FIG. 7) to evaluate the ability' of the combined vaccine to protect against repeated tick infestations.

[9081] The foregoing study begins with rabbit models and proceeds to mouse and then white-tailed deer models. However, in other implementations, the study can begin with the mouse model as outlined to test the tick bacterial endosymbiont R. buchneri for its potential efficacy as: an anti-tick vaccine. Specifically, the study determines whether whole R. buchneri cells are immunoreactive in a laboratory mouse model and followed by a challenge of mice with I. scapularis ticks to quantify any negative effects on the ticks (i.e., mortality, low rate of weight increase, and / or changes in feeding duration) during blood feeding.

[0082] R. buchneri can be grown in tick cell culture using existing methods. After growing R. buchneri in I. scapularis ISE6 cell culture, live bacteria are released from the eukaryotic 1SE6 cells. Whole J?, buchneri cells can then either be used intact (heat killed) or lysed in preparation for mouse vaccinations. The target immunization for each mouse is the equivalent of approximately lxl06i?. buchneri cells. The number of R. buchneri cells is estimated using a real-time polymerase chain-reaction (PCR) assay to quantify a single copy locus from the 2?. buchneri core genome. The same locus is placedin a synthetic “G-block” DNA strand that serves as a PCR control with a known copy number (1 copy per synthetic G-block). DNA from the R. buchn eri whole-cell preparation is diluted serially and analyzed in multiple PCR replicates, which can be compared against the G-block control to obtain a relative quantification of the number off?, buchneri core genome copies. The number of genome copies is directly correlated with the number off?, buchneri cells per given volume,

[0083] In advance of the mouse study, a verification is conducted on the laboratory colony off. scapularis ticks to prove that they actually cany the R. buchneri endosymbiont. This is done by extracting DNA from a sample ofl. scapularis ticks from the lab colony and using the same real-time PCR assay described above.

[0084] Ten experimental mice (5 females and 5 males) can be given a subcutaneous immunization with the equivalent of approximately lxl06i?. buchneri cells in lx PBS using TITERMAX GOLD adjuvant; six control animals (5 females / 5 males) will receive only lx PBS with TITERMAX GOLD adjuvant (10 mice total). The experimental timeline is as shown in FIG. 8; a single booster is given on day 21. Blood samples (<100 microliters) from each mouse will be collected at five timepoints to quantify' total IgG titer to each antigen. Sera monitoring continues during the experiment to determine if vaccinated animals raise a significantly higher level of IgG antibody compared to controls;a suitable cutoff point is an OD 450 nm reading of 0.35 for a 1 / 500 dilution of serum. Each mouse can then be challenged with ticks to evaluate any negative effects to the ticks. At the end of the experiment, mice will be euthanized and serum collected from a final blood draw (1-5 mL per mouse). On day 31, each mouse is challenged with the nymph stage of / . scapularis (n=20 ticks per mouse). Host animals are prepared for infestation by shaving hair from a small dorsal area of the thorax and gluing pouches to the skin. Ticks are introduced via the resealable pouch opening and allowed to feed until they engorge and / or drop from the host, about 4 days for typical nymphs. Detached ticks are removed daily and weighed. Ticks will be incubated at 22 C under 85% humidity in a 16:8 hr light' dark cycle to evaluate the molting rate of nymphs. This incubation takes about 2 months and is a good predictor of vaccine efficacy.[0085| Data collected during the study includes total IgG ELISA. The sera collected at 1-2 week intervals are analyzed with ELISA to evaluate total IgG responses. Additional data includes mouse skin reactions at the bite site (photos) and spleens will be saved to attempt recovering antibodies specific to individual R. buchneri OMPs (i.e., OmpB, BamD, etc.). The effects of tick feeding from vaccinated vs control mice with the statistical analysis of the following measurements is also calculated including tick mortality, tick feeding duration, tick weight upon detachment, and the molt rate of nymphs. Any impacts to the R. buchneri symbionts are also assessed by extracting DNA from 7. scapularis nymphs upon completion of the study. Using these DNA samples, the real-time PCR described above (including the synthetic G-block) is used to perform relative quantification of the J?, buchneri symbiont in whole ticks after feeding on immunized vs. control hosts.

[9086] Additional testing can be carried out including screening against whole-cell lysates of R. buchneri in Western blots to confirm total IgG antibody reactivity' withendosymbiont proteins. This analysis includes Western blots containing several types of negative controls. Pre-vaccination mouse serum (day 0) is screened against whole-cell lysates of R. buchneri in Western blots to examine the expected lack of IgG antibodies that are reactive with endosymbiont proteins. Serum from non-vaccinated control mice is screened against whole-cell lysates of R. buchneri in Western blots to examine the expected lack of IgG antibodies binding to endosymbiont proteins. Eukaryotic cell lysate from pure ISE6 tick cell culture is prepared to reveal if any tick proteins from cell culture inadvertently contaminated the R. buchneri whole cells during the isolation procedure and thus were included in the vaccine formulation. Single whole-length recombinant protein (i.e., OmpB) is tested against mouse sera using ELISA to evaluate specific IgG reactivity. For ticks that show obvious external signs of distress (discoloration, bloating), microscopy of the midgut, is used to identify gross negative effects. Microscopy using immunofluorescence assay (IF A) to image antibody atachment on the outer membrane of R. buchneri cells is also used. Live, intact cells of?. buchneri isolated from ISE6 cell culture is exposed to mouse antibodies. If mouse antibodies are capable of attaching to endosymbiont OMPs they may neutralize the activity of these proteins. If neutralization is highly effective, the R. buchneri cells can be expected to have a decreased ability to enter uninfected ISE6 tick cells. This can be tested by adding antibody-treated R. buchneri cells to a culture of live ISE6 cells and looking at the entry rate of tick cells through time. This is then compared to a baseline control that uses R. buchneri cells not exposed to antibodies that wall retain their ability to enter ISE6 tick cells at a high rate.

[0087] The main outcomes of the study are 1) ELISA data from IgG antibodies evaluating the immunogenicity of R. buchneri cells, 2) data to determine if vaccination reduces I. scapularis feeding efficiency (i.e., feeding time and weight) and reproductive fitness of females (i.e., egg mass weight and hatching success). Because bacterialendosymbionts are essential for tick bloodmeal processing, immunization with R. buehneri whole cells should have a strong chance of being protective against ticks. Success criteria for the study include any or all of the following: significant tick mortality after feeding on vaccinated hosts versus control hosts: significantly lower mean weight of ticks after feeding on vaccinated hosts versus control hosts: any statistically significant changes to tick feeding behavior, such as early detachment or prolonged feeding that are significantly different from ticks fed on vaccinated hosts versus control hosts; an ELISA reading (OD 450 nm) that is significantly greater in the serum of vaccinated hosts versus control hosts; a reduction in the quantity of R. buehneri cells remaining in ticks that fed on vaccinated hosts versus control hosts; a reduction in the rate at which antibody-treated R. buehneri cells can enter live ISE6 tick cells through time, as compared to a baseline control using untreated R. buehneri cells added to ISE6 tick cells.[0088 The foregoing analy ses have applied to J. scapukiris ticks. Similar applica tion of the principles disclosed can be applied to those ticks that carry cattle fever (cattle fever ticks like any previously disclosed). Because the CLERM proteins are not as well understood, the analysis is laid out in FIG. 9 where a first step is to create a prioritized list of CLERM protein targets. The selection criteria may include, by non-limiting example, proteins encoded by the core genome, outer membrane proteins, proteins predicted to have highly immunogenic B-cell epitopes, current vaccine candidates against Coxielki burnetii, or highly conserved proteins found in other CLE species in other ticks. With this prioritized list of CLERM protein targets amino acid variation within 20 of these proteins will be examined across a large R. microplus / CLERM dataset (n=960) using AmpSeq. AinpSeq is a PCR-based, high-throughput sequencing approach facilitating inexpensive, multiplexed sequencing at many genomic locations (several hundred) across multiple samples simultaneously. Because the output is nucleotide sequences from DNAtemplates, it provides a means to identify both known and unknown mutations at genomic regions of interest For example, it can be leveraged to assess certain mutations (i.e,, single nucleotide polymorphisms (SNPs)) associated with specific strains, species, or phenotypes. Multiplexed PCRs for each sample are normalized and sequenced, providing hundreds to thousands of reads per PCR target, and reads are then aligned to a reference using the Amplicon Sequence Analysis Pipeline ( ASAP). The outcome of the AmpSeq analysis is information on protein allele variants for the about 20 proteins being considered to be included in a vaccine composition.[0089J With about 5 top CLERM proteins identified from the previous analysis, the ability of the CLERM proteins to induce an IgG antibody response in cattle is assessed as illustrated in FIG. 10. The use of recombinant proteins rather than native proteins from cultured cells may be used because of the difficulty in culturing CLEs outside of their hosts. As illustrated in FIG. 10. a second round of vaccinations and tick trials is carried out in catle using bivalent formulations that contain both the selected CLERM protein(s) and, in this implementation, the tick VDAC protein, which has high efficacy against tick infestation. This work will provide a foundational understanding of the immunogenic potential of the selected CLERM proteins in concert with a tick protein and evaluate efficacy of R. microplus control on cattle. Importantly, because CLEs like or related to CLERM are widespread across many tick species, and often ubiquitous within them, a CLERM-based vaccine (or a vaccine using a conserved protein from any CLE type disclosed herein) have great potential as a tool to control multiple tick species. This includes other tick species with CLEs that also transmit livestock pathogens, including R. annulatus and H. longicorms, and ticks that transmit important human pathogens, such as R. sanguineus, which transmits Rickettsia ricketlsii (Rooky Mountain Spoted Fever) and other pathogens to humans and animals.1 0901 The process of selecting the CLERM proteins is provided in greater detail in the diagram illustrated in FIG. 11. Starting from the CLERM core genome a prioritized list of anti-CLERM protein vaccine targets is created using the process summarized in FIG. 11. First, proteins are considered which are encoded by the CLERM core genome. All intact proteins encoded by genes present in the CLERM core genome are first identified. As pseudogenes can accumulate during CLE evolution, genes encoding for partial length proteins are filtered out to ensure only full-length open reading frames are retained for consideration.0091| Outer membrane proteins encoded by the CLERM core genome are then identified along with modeling protein folding and 3D structure using AlphaFold 1 to identify ceil surface domains. Epitope prediction tools are then used (IEDB131, ABCpredl32, PSORTbl28, and VaxiJenll3 to identity CLE OMPs that likely contain highly immunogenic B-cell epitopes. Epitopes are then mapped onto 3D models to prioritize those that are surface exposed. As a quality control step, CLE proteins that have low allergenicity values for cattle are then screened and selected.

[0092] As part of selection, prioritizing vaccine candidates currently under development against C burnetii that are also found in the CLERM is also done. Leading C. burnetii vaccine targets include proteins such as OmpA, OmpH. YbgF, and translation elongation factor Tu, all of which are encoded in the CLERM genome and share reasonable levels of amino acid identi ty with C. burnetii. During the selection process, a comparison with available genomes of CLEs from other tick species to the CLERM genomes in the study is carried out to evaluate conservation of target proteins across CLE species. This will help identify targets that may be useful against multiple tick species.

[9093] After doing the foregoing, a prioritized list of about 20 proteins to screen across a large (n=960) R. micropIus CLERM DNA collection using AmpSeq is thenselected. The resulting data provides an essential understanding of variation in the CLERM protein candidates across diverse 2?. microplus populations in different geographies in the US and around the world. In this analysis, the best five CLE proteins will be moved along to cattle / tick trials.

[0094] The next step is to test the five best CLERM OMP candidates for their ability to raise a strong IgG antibody response in a bovine model and protect against tick infestations. The vaccination trials in cattle that will allow us to: 1 ) compare antigenicity across individual CLERM proteins, and 2) evaluate the ability' of single CLERM anti gens combined with the VDAC4 tick antigen to protect vaccinated cattle against ticks.Recombinant E. coli expression plasmids that encode the CLERM proteins and a histidine tag (his6 ) on the N-tcnninus of each protein using a cloning system marketed under the tradename GATEWAY by ThennoFisher of Waltham, Massachusetts. Proteins are expressed in E. coli Rosetta cells from Millipore Sigma, lysed, and purified using a nickel affinity column marketed under the tradename HISPREP FF 16 / 10 by GE Life Sciences of Marlborough, Massachusetts: the latter captures any proteins via the his6 tag. Bound proteins are eluted using an imidazole concentration gradient. Eluted fractions containing the protein of interest are identified by SDS-PAGE and pooled, dialyzed, quantified with a BCA assay, and confirmed with Western blot analysis using a monoclonal antibody specific to the his6 tag. Because the analysis focuses on CLERM OMPs (OmpA, etc.), some or all of the target proteins may be insoluble. Insoluble proteins may be successfully purified by adding an E. coli thioredoxin solubility tag paired with the his6 tag, or by chemical solubilization using N-Lauroylsarcosme. Proteins purified using both methods are amenable for mammal immunization.

[9095] In the first round of vaccine / tick trials and referring to FIG. 12, five individual CLERM recombinant proteins’ ability to stimulate an IgG antibody response incattle and protect against experimental tick infestation is evaluated. Each of the five antigens will be tested separately (sequentially, not parallel) in groups of five experimental cattle (Bos taunts outbred European breeds), for a total of 25 experimental cows plus one set of five control cows for comparison to all experimental groups (30 animals total in trial 1). To form the vaccine, 100 micrograms of purified recombinant protein is mixed in 1 mL of a VG adjuvant marketed under the tradename MONTANIDE ISA 201 by Seppie of ColOmbes, France. This adjuvant is recognized for its ability to rapidly stimulate an IgG response in cattle. The vaccination / tiek challenge experiment will take up to 70 days as indicated in FIG. 12, with initial vaccination on Day 0 and a booster on Day 21. Larval ticks of At microplus are added 10 days later (Day 31) to coincide with high IgG levels. Adult females are collected after engorgement (Day 52+); engorgement is anticipated to take longer for ticks fed on vaccinated hosts and this timeline accounts for this. At multiple timepoints, 10 mL blood samples are collected as illustrated in FIG. 12 for antibody testing with ELISA.

[0096] F or tick infestations on cattle, 0.5 g of larval R. microplus (—10,000 ticks) from a specified lab colony are separated into two cloth chambers glued to shaved skin patches. Ticks remain on cattle until adult females are engorged (~3 weeks), at which time females that drop from cattle will be collected daily (-21-25 days post infestation).Multiple parameters of female tick fitness in control versus, vaccinated groups are collected during this time, including the number of females that survive, weight, egg mass weight, egg hatching success, larval weight, and larval survival. All live engorged female ticks (typically <1,000 for a control) will be disinfected, counted, weighed, and placed in petri dishes to oviposit for thirteen days at 26 C and 80% humidity'. The total combined tick egg mass collected from each single cow being tested will be collected and weighed. Egg viability is then estimated using 0.2 g of total egg mass from each bovine, combined acrossthe five cows in each group (i.e., 1,0 g total per group), with triplicate samples from each cow. Eggs are then incubated until 15 days after larval groups hatch and the number of unhatched eggs remaining will be estimated by weight. A 0.1g sample of larvae (-2,000) is counted and sorted as alive or dead to evaluate early survival. We will then perform statistical comparisons between control versus vaccinated groups to evaluate the effect on the ticks throughout. Finally, vaccine efficacy is calculated using a standard formula that is widely implemented in cattle fever tick studies, which incorporates the number of surviving females, egg mass w’eight, and larval weight per gram of eggs.

[0097] As part of the testing, a quantification of IgG antibody responses is carried out. The immunogenicity of each CLERM protein is evaluated using ELIS A containing the purified protein and cattle-specific secondary antibodies to quantify protein-specific IgG levels in vaccinated versus control animals (n=5 in each group). ELISAs are also used to quantity IgG levels of CLERM and tick proteins used in subsequent bivalent vaccine trials discussed later. This immunological data provides key information on the capacity of CLERM proteins to stimulate an IgG antibody response that protects against ticks.

[0098] To perform Western blot analyses and verify tissue localization, intact midgut with Malpighian tubules is harvested from 30 adult female ticks (not used in the vaccination experiment) from the lab colony. The tissues are then placed into two pools, flash-frozen in IxPBS, and shipped for laboratory analysis, where one pool is used to make tissue homogenates and the other used for localization studies. Antibodies from vaccinated cattle sera are then used to confirm the presence of the final five target CLERM proteins in the homogenized tissues via Western blot analysis. Antibodies also are used to perform immunochemical staining of intact tissues to verify that: 1) the targeted VDAC and CLERM proteins are indeed present and expressed in the expected locations, and 2) IgG antibodies are able to access and react with target proteins in situ. This is an important stepfor validating protein targets and evaluating reactivity with IgG antibodies. Finally, DNA will be extracted from a small amount of tissue to test with the CLERM-specific qPCR assay previously discussed to broadly assess CLERM abundance.

[0099] During the challenge experiments, adult female ticks are collected and surface disinfected from control and vaccinated animals (n=10 ticks per cow; 50 ticks per group of five cows) to extract DNA and perform 16S community analysis, as described previously. This approach is used to reveal broad effects on tick microbiome composition in adult females that result from specific IgG antibodies reacting with OM proteins of the obligate endosymbiont. Insight into the relative contribution of each CLERM target is also obtained by making comparisons across the five vaccinated groups. Because the CLERM is vertically transmitted from females to eggs and thus to larvae, DNA is extracted from pooled egg mass samples and pooled larval samples to perform real-time PGR for relative CLERM quantification using the real-time PCR assay previously described. With this analysis, the amount of CLERM in eggs and larvae is evaluated to determine if it is reduced after females feed on vaccinated bovine hosts.[00100 j Following completion of the first trial experiment, the efficacy of bivalent vaccines containing at least one CLERM protein and the tick VDAC protein will be evaluated. Two bivalent vaccines will be tested in groups of five cattle and one control group of five cattle (15 animals total in trial 2). The two CLERM proteins utilized in the second trial are selected based upon their efficacy in trial 1. This trial will follow the same methods as trial 1 summarized in FIG. 12, including evaluating tick fitness and estimating vaccine efficacy.

[0101] The genomic analysis of the R. microplus tick vaccines arid the corresponding CLERM endosymbiont proteins also includes conservation analysis based on an extensive database of geographically dispersed tick collections over a significantperiod of time that include tools that allow for accurate data collection. Among these tools are microsatellite-based genotyping systems for 7?. microplus and R. annulatus and additional assays, to detect genetic mutations associated with resistance to several classes of acaricides. Applying these tools has important insights about CFTs in Texas and. Mexico, including: 1 ) CFT infestations are shared freely between wildlife and cattle, 2) shortdistance tick dispersal typically results from infested wildlife movements, whereas longdistance dispersal always results from transport of infested cattle, and 3) resistance mutations for synthetic pyrethroids frequently spread from Mexico into Texas. Because of this long-term partnership with USDA and TAHC, and also with other CFT scientists, an extensive collection of DNA extracts from >8,000 individual R. tnicraplus ticks is used the conservation analysis. The majority are from geographically diverse field collections in Texas and Mexico as illustrated in FIG. 13, but DNA from CFT collections from Brazil, Colombia, and Puerto Rico is also included. Importantly, all DNA extractions utilized whole or half tick bodies and, thus, include DNA from the entire microbiome community, including the CLERM, Because these extracts have already been genotyped, representative subsets for various conservation studies can be prepared that are both genetically and geographically diverse.

[0102] The need for carefill evaluation of the conservation of the genes that encode targeted endo symbiont proteins across multiple representatives of that species is illustrated by the diagrams in FIG. 14. On the left the shared genes (translating into shared proteins) between three different CLERS genomes collected from brown dog ticks are represented by circles with the intersecting regions containing the number of shared genes between the genomes. This diagram indicates that while most of the genes are shared, there are a significant number that are not shared at all between genomes. Thus, ensuring that the analyses only include genes / proteins actually shared among CLERS genomes is animportant filtering step to ensure that vaccine escape does not happen. In the diagram on the right, the genes from a CLERM genome, a CLERS genome, and from CoxieMa burnetii are each displayed using a circle with the total common genes represented by the overlapping regions. Notwithstanding the multispeeies comparison, a majority of the genes in each genome are common, which indicates that a common conserved protein between the three species could be used to create a vaccine that would attach to each bacterium relatively equally and thus increase the universality of the vaccine.

[0103] One of the strategies for endosymbiont protein selection involves considering proteins involved in the shikimate pathway. As previously mentioned, the shikimate pathway has been observed in the Asian longhomed tick and through this pathway, the endosymbiont produces the serotonin that the tick responds to that creates the interest and desire to feed on the host. The paper to Zhong previously incorporated by reference demonstrated that when the endosymbiont C’LEHL was killed using tetracycline, the feeding behavior of the ticks changed causing them to not have the desire to feed. The inability to feed can significantly impact female ticks as, while they may not die and drop themselves as a result, their ability to create and lay eggs may be significantly hindered or eliminated meaning that no new larval ticks can be created in the next generation. Thus, where the shikimate pathway is known to exist in a tick endosymbiont, utilizing a protein that causes IgG antibodies to significantly reduce the total number of bacteria may have the effect of causing the ticks to starve even if they are otherwise able to process blood meals.

[0104] The shikimate pathway exists in various tick endo symbionts. It is believed to be conserved in the CLERM based on analysis of 23 CLERM genomes which recovered 721 genes of this endosymbiont wi th 669 genes found in all 23 CLERM genomes. While the aroD protein in Francisella tularensis is not homologous to the gene in CLE, a type 13-dehydroquinate dehydratase-, it is believed that sufficient genes exist in the CLERM for theshikimate pathway to exist. With respect to the CLERS, the genetic assembly is missing the shikimate dehydrogenase gene aroE and subsequent enrichments are also missing this gene which possibly indicates that a fell sliikimate pathway is not present in CLERS. With respect to the CLEAA, the shikimate pathway is believed to exist, but a similar issue with the aroD is present as with the CLERM. An analysis of.9 adult female ticks that included genome enrichment to capture the transcriptome of CLERM identified 6 outer membrane proteins that were expressed in all 9 ticks, indicating conservation that means these could be used as vaccine target proteins,[00105| In various vaccine implementations, the tick protein and endosymbiont protein can be selected to enable multispecies effectiveness. For example, a combined vaccine administered orally using baits that targets both I. scapu aris and A. americanum and their corresponding endosymbionis could be constructed. This combined vaccine would target the carriers of the most important vector-borne disease in the United States, Lyme disease and the increasing problem of alpha gal previously discussed. Individual landowners would buy oral baits and leave them out on their properties to treat the local deer and mouse populations to correspondingly reduce local tick populations and simultaneously reducing the risk of tick feeding and pathogen transmission to humans,

[90106] In another implementation, a combined vac erne composition in an injectable form, or deliverable to mucosal surfaces through other mechanisms such as a gene gun, could be prepared that targets D, vanablis / D. similis, I. scapularis,.4. americanum, and / or R. sanguineus and all of their respective endosymbionts could be developed using the principles disclosed herein. This vaccine would cover all of the most common ticks found on pet dogs and cats in the United States. The vaccine could be given during routine veterinary examinations like the rabies vaccine. This vaccine has the similarbenefit of protecting humans from these ticks as well as the dogs and cats from pathogens transmitted from these ticks.

[0107] The following references are each hereby incorporated entirely herein by reference:

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[0162] In places where the description above refers to particular implementations of anti-tick vaccine compositions and related methods and implementing components, subcomponents, methods and sub-methods, it should be readily apparent that a number of modifications may be made without departing from the spirit thereof and that these implementations, implementing components, sub-components, methods and sub-methods may be applied to other anti-tick vaccine compositions and related methods.

Claims

CLAIMSWhat is claimed is:

1. An anti-tick vaccine composition comprising:one or more conserved tick proteins from a tick species; andone or more conserved outer membrane proteins of an endosymbiont of the tick species.

2. The composition of claim 1, wherein the one or more conserved tick proteins and the one or more conserved outer membrane proteins each have a conservation level equal to or greater than 97%.

3. The composition of claim 1. wherein the tick species is Rhipieephalux microplus and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 1-28 and any combination or fragment thereof and the endosymbiont is CLERM and the one or more conserved outer membrane proteins is selected from the group consisting of SEQ ID NO: 190-207 and any combination or fragment thereof.

4. The composition of claim I, wherein the tick species is Rhipieephalux sanguineus sensu lato and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 29-56 and any combination or fragment thereof and the endosymbiont is CLERS and the one or more conserved outer membrane proteins is selected from the group consisting of SEQ ID NO: 221-232 and any combination or fragment thereof.

5. The composition of claim 1, wherein the tick species is Amblyomma americaniim and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 113-440 and any combination or fragment thereof and the endosymbiont is CLEAA and the one or more conserved outer membrane proteins is selected from the group consisting of SEQ ID NO: 233-242 and any combination or fragment thereof.

6. The composition of claim 1, wherein the endosymbiont is CLERA and the one or more conserved outer membrane proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 208-220 and arty combination or fragment thereof.7, The composition of claim 1, wherein the tick species is Ixodes scaptdaris and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 57-84 and any combination or fragment thereof.

8. The composition of claim 1, wherein the tick species is Ixodes pacific us and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 85-112 and any combination or fragment thereof.

9. The composition of claim 1, wherein the tick species is Haemaphystd is laiigicornts and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or100% identical to a protein selected from the group consisting of SEQ ID NO: 141-161 and any combination or fragment thereof.

10. The composition of claim 1, wherein the tick species is Dermacenlor variabilis and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 162-189 and any combination or fragment thereof11. A method of vaccinating an animal against hosting a tick, the method comprising:administering to the animal a pharmaceutically effective amount of a bivalent vaccine comprising one or more conserved tick proteins from a tick species and one or more conserved outer membrane proteins of an endosymbiont of the tick species.

12. The method of claim 11, wherein administering comprises injecting or delivering the vaccine into a body or mucosa of the animal or oral application to the animal and where the bivalent vaccine further comprises where the one or more conserved tick proteins and the one or more conserved outer membrane proteins each have a conservation level equal to or greater than 97%.

13. The method of claim 11, wherein the tick species is Rhipicephalus microplus and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 1-28 and any combination or fragment thereof and the endosymbiont is CLERM and the one or more conserved outer membrane proteins is selected from the group consisting, of SEQ ID NO: 190-207 and any combination or fragment thereof14. The method of claim 11, wherein the tick species is Rhipicephalus sanguineus sensu lato and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 29-56 and any combination or fragment thereof and the endosymbiont is CLERS and the one or more conserved outer membrane proteins is selected from the group consisting of SEQ ID NO: 221-232 and any combination or fragment thereof.

15. The method of claim 11, wherein the tick species is Amblyomma americanum and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100%) identical to a protein selected from the group consisting of SEQ ID NO: 113-140 and any combination or fragment thereof and the endosymbiont is CLEAA and the one or more conserved outer membrane proteins is selected from the group consisting of SEQ ID NO: 233-242 and any combination or fragment thereof.

16. The method of claim 11, wherein the endosymbiont is CLERA and the one or more conserved outer membrane proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 208-220 and any combination or fragment thereof.

17. The method of claim 11, wherein the tick species is Ixodes scapularis and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90 ), 95%, of 100% identical to a protein selected from the group consisting of SEQ ID NO: 57-84 and any combination or fragment thereof.

18. The method of claim 11, wherein the tick species is Ixodes pacificus and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 85-112 and any combination or fragment thereof.

1. The method of claim 11, wherein the tick species is Haemaphysatis longicomis and the one or more conserved tick proteins is at least 75%, 80%, 85%. 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 141-161 and any combination or fragment thereof.20, The method of claim 11, wherein the tick species is Dermacentorvariabilis and the one or more conserved tick proteins is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to a protein selected from the group consisting of SEQ ID NO: 162-189 and any combination or fragment thereof.

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