WO2024069028A1

WO2024069028A1 - Vaccine for preventing infection with anaplasma phagocytophilum (rickettsiales: anaplasmataceae)

Info

Publication number: WO2024069028A1
Application number: PCT/ES2023/070565
Authority: WO
Inventors: Marinela CONTRERAS ROJO; José de Jesús DE LA FUENTE GARCÍA
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2022-09-30
Filing date: 2023-09-21
Publication date: 2024-04-04
Also published as: ES2967368A1

Abstract

The invention relates to a chimeric protein containing peptides that protect against infection by the pathogen Anaplasma phagocytophilum. The chimeric antigen comprises epitopes of Anaplasma phagocytophilum MSP4 protein, specifically four MSP4 amino acid subsequences. The chimeric antigen produced a greater immune response in comparison with the administration of the complete MSP4 protein, and with a control peptide when same was administered to animals susceptible to Anaplasma phagocytophilum infection. The chimeric antigen therefore is advantageous for the prevention and/or treatment of infections caused by bacteria of the Anaplasmataceae family.

Description

DESCRIPTION

Vaccine for the prevention of Anaplasma phagocytophilum infection

(Rickettsiales: Anaplasmataceae)

The present invention is found within the biotechnology, biochemical and pharmaceutical sectors. The present invention relates to a recombinant chimeric antigen that can be used for the prevention or treatment of infections caused by Anaplasma phagocytophilum.

BACKGROUND OF THE INVENTION

Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) is an emerging tick-borne intracellular bacterial pathogen in many regions of the world that causes human granulocytic anaplasmosis (HGA), tick-borne fever (TBF), and canine anaplasmosis. A. phagocytophilum infection has been documented to infect a wide range of hosts, including cattle, sheep, goats, dogs, horses, humans, and red deer (Cervus elaphus). , the roe deer (Caperolus capreolus) and the white-tailed deer (Odocoileus virginianus) and several rodents (Woldehiwet, Z. 2010. Vet. Parasitol. 167, 108-122; Stuen, S., et al. 2013. Front Cell Infect Microbiol. 3, 31).

In humans, doxycycline hypochlorite is the agent of choice for the treatment of anaplasmosis with clinical improvement within 24-48 h and cure. In other species, the prophylactic use of tetracycline together with the applications of acaricides, to control ticks, are the main measures to control A. phagocytophilum infection in endemic areas. However, these control measures raise concerns about their impact on the environment and human health, and the selection of resistant pathogens and ticks (Bakken, J. S. and Dumler, J. S. 2006. Ann N Y Acad Sci. 1078, 236-247; Maurin, M., et al. 2003. Agents Chemother. 47, 413— 415).

At the moment, there are no vaccines available for the prevention and control of A. phagocytophilum, however, the vaccines that could be developed would be aimed at affecting the tick, the pathogen it transmits, or both to control anaplasmosis. The use of vaccines would also include the advantage of avoiding contamination environmental protection and the selection of pesticide-resistant arthropod vectors, while improving animal welfare, preventing the transmission of ticks and pathogens for tick-borne diseases. In the control of these pathogens, the tick protective antigen Subolesin (SUB) appears to be a candidate antigen that can contribute to the control of multiple species of ticks and as a consequence reduce the transmission of pathogens transmitted by them (de la Fuente J., et al. al. 2006. Parasitol Res 100:85-91).

One of the main limitations to the development of effective vaccines for the prevention and control of A. phagocytophilum infection and transmission is the identification of effective protective antigens. Studying molecular interactions between ticks and transmitted pathogens would enable the identification of candidate tick antigens to reduce infection and pathogen transmission, while also affecting tick infestations. Recent results have shown that the application of a vaccinomic approach to the interactions between the tick Ixodes scapularis and A. phagocytophilum allowed the identification and characterization of candidate protective tick antigens for the control of vector infestations and A. phagocytophilum infection ( Contreras M., et al. 2016. Methods in Molecular Biology, vol. 1404, 275-286; Contreras M., et al. 2019. Front. Physiol. 10:977)

In the same way, studying the proteins involved in the strategies by which A. phagocytophilum establishes the infection, it has been found that the major surface protein 4 (MSP4) located in the bacterial membrane, plays a possible role during the infection of the pathogen in ticks (Villar, M., et al. 2015. PLoS ONE 10:e0137237) and would be a possible vaccine target, characterizing its potential protective capacity in immunized animals. However, previous vaccination trials in sheep with the MSP4 antigen of A. phagocytophilum (Contreras M., et al. 2017. Front. Cell. Infect. Microbiol. 7:307) showed only partial protection in lambs and differences between antibody responses of rabbits and sheep. Thus, there is a need to provide new vaccination strategies that allow the development of a protective response against A. phagocytophilum.

DESCRIPTION OF THE INVENTION In previous vaccination trials (Contreras M., et al. 2017. Front. Cell. Infect. Microbiol. 7:307) in sheep with the MSP4 antigen (with accession number in GenBank: AFD54597) of A. phagocytophilum showed differences between the antibody responses of rabbits and sheep, which may be associated with the recognition of non-protective epitopes by the IgG of immunized lambs and protective epitopes by the IgG of immunized rabbits.

The inventors have developed a chimeric antigen comprising the sequence SEQ ID NO: 1, hereinafter "chimeric antigen of the present invention", by identifying and characterizing the protective epitopes of MSP4 against A. phagocytophilum, since the Use of the full MSP4 protein does not induce a complete protective response.

The inventors performed a search for epitopes of the MSP4 protein using a vaccinomics-based approach. The analysis of the distinctive reactive epitopes showed that the protective IgG antibodies from rabbits immunized with MSP4 recognized 4 epitopes or immunogenic regions of said protein (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5). Therefore, the chimeric antigen of the present invention was constructed from the identification of these 4 epitopes of the MSP4 protein of A. phagocytophilum. Therefore, the chimeric antigen constructed by the inventors comprises the immunogenic peptides (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5) identified by rabbit protective IgG antibodies. Furthermore, the inventors also identified by the same approach an MSP4 peptide recognized by IgG antibodies from sheep immunized with MSP4, but which turned out to be non-protective (SEQ ID NO: 6).

The antigen object of the present invention designed by the inventors comprises the following sequence:

SEQ ID NO: 1:

Said antigen comprises the peptide sequences:

SEQ ID NO: 2:

The peptide used as a negative immunization control comprises the sequence:

Subsequently, an in vitro assay was performed in which purified IgG antibodies from rabbits and lambs immunized with A. phagocytophilum MSP4 were used to characterize the inhibition of infection by the pathogen in HL60 and ISE6 cells in comparison with the protective capacity. of the IgGs from sheep immunized with the new chimeric antigen of MSP4 (SEQ ID NO: 1) and a non-protective peptide in sheep as a negative control (SEQ ID NO: 6) obtained from the analysis of the MSP4 microarray. Surprisingly, it was found that IgG from sheep immunized with the chimeric protective peptide object of the invention produced a greater inhibition of infection by the pathogen A. phagocytophilum in HL60 and ISE6 cells than IgG from sheep immunized with the peptide SEQ ID NO: 6 and IgG from lambs immunized with MSP4 did not protect against A. phagocytophilum infection (Fig. 3).

In a first aspect, the present invention relates to a chimeric protective antigen against A. phagocytophilum comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 1; where the sequence SEQ ID NO: 1 comprises the amino acid sequences of the peptides SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 which belong to the amino acid sequence of the protein MSP4 from A. phagocytophilum. The term “peptide,” as used herein, includes both the full-length protein and shorter polypeptide and peptide sequences.

In the context of the present invention the term "amino acid sequence" or "polypeptide sequence", used interchangeably, refers to the linear sequence of amino acids joined by peptide bonds that make up a peptide or protein.

In the present invention, the term "protective against" or "immunogenic" refers to the ability of a molecule to generate a protective immune response against a pathogenic organism, that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that sterilize or reduce the reproduction rate of the invading organism or damage, inhibit or kill it, thus “protecting” the host from clinical or subclinical disease and loss of productivity. Such a protective immune response can commonly manifest itself by the generation of antibodies that are capable of inhibiting the metabolic function of the invading organism, leading to an impediment to its normal growth, lack of reproduction and/or death. The term "immunogen" refers to a molecule capable of generating a response as described above, in the present invention an immunogen can be a peptide, polypeptide, protein, fusion protein, chimeric protein or chimeric antigen.

The term “antigen” in this document refers to a molecule that can induce the formation of antibodies thereby generating a protective response. There are many different types of molecules that can act as antigens, such as proteins or peptides, polysaccharides and, more rarely, other molecules such as nucleic acids. In the context of the present invention the antigen is a molecule selected from the list comprising protein, peptide, polypeptide, fusion protein or chimeric protein. The term "antigenic" refers to the characteristic or ability of a molecule, according to the present invention, to induce the formation of antibodies.

According to the description, the term "chimeric antigen", "chimeric protein" or "fusion protein", used interchangeably, is understood as a peptide or protein whose amino acid sequence comprises two or more amino acid sequences or peptides of the same or several proteins, where the sequence Polypeptide of the “chimeric antigen”, “chimeric protein” or “fusion protein” does not occur in a single molecule in nature, or at least the sequences that compose it are not linearly contiguous in a single molecule in nature.

In the context of the present invention, the term "chimeric antigen", "chimeric protein" or "fusion protein" of the present invention, terms used interchangeably, is understood to mean the sequence of amino acids that forms the peptide, polypeptide or protein object of the present invention, where said sequence comprises the amino acid sequence SEQ ID NO: 1 and is capable of generating an immunogenic or antigenic response against A. phagocytophilum.

The inventors observed that the peptides with sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 make up epitopes of the MSP4 protein that were specifically recognized by IgG from rabbit and lamb sera. previously vaccinated with the MSP4 recombinant protein.

In a preferred embodiment, the chimeric antigen of the present invention is linked by one of its N or C terminal ends to an amino acid sequence of a bacterial membrane anchoring protein or protein fragment. Said bacterial membrane anchor protein or protein fragment allows peptides to be expressed in the membrane of a host cell for subsequent isolation and purification. Preferably the amino acid sequence of a bacterial membrane anchoring protein or protein fragment has an identity of at least 80%, 90%, 95% or 98% with the amino acid sequence SEQ ID NO: 7, more preferably where said amino acid sequence comprises, even more preferably consists of, the sequence SEQ ID NO:7. This sequence corresponds to a fragment of the MSP1 a protein from Anaplasma margínale. The MSP1 a protein (Major Surface Protein 1 a) is one of the five known major surface proteins of A. margínale and is involved in the adhesion of the pathogen to hosts and in interactions of the pathogen with ticks. .

SEQ ID NO: 7:

The sequence SEQ ID NO: 7 corresponds to a portion or fragment of the MSP1 a protein of A. marginale to direct the exposure of other peptides on the cell surface, through its N-terminal fusion with this protein. Taking into account the natural size range of the repeat peptides in the N-terminal region of MSP1 a (28-289 amino acids) and the exemplary embodiments set forth herein, an advantage of using the MSP1 a protein instead of other motifs anchoring membrane proteins to expose peptides on the cell surface, is the possibility of exposing polypeptides of different sizes and amino acid composition. In the present description SEQ ID NO: 7 is referred to as MSP1 mutant protein a. The chimeric antigen with SEQ ID NO: 1 is preferably linked to the N-terminal region of the MSP1 a protein fragment (SEQ ID NO: 7).

In a preferred embodiment the chimeric antigen of the present invention comprises, preferably consists of, the amino acid sequence SEQ ID NO: 8.

SEQ ID NO: 8:

The sequence SEQ ID NO: 8: corresponds to a polypeptide or fusion protein that comprises the chimeric antigen made up of the antigenic fragments or peptides of MSP4 (SEQ ID NO: 1) with a methionine residue at the N-terminal end, fused to the MSP1 a protein fragment (SEQ ID NO: 7), where the sequence of the chimeric antigen SEQ ID NO: 1 is attached to the N-terminal end of the MSP1 a protein fragment (SEQ ID NO: 7).

A second aspect of the invention relates to a nucleotide sequence encoding the chimeric antigen according to the first aspect of the invention, wherein the nucleotide sequence comprises, preferably consists of, the nucleotide sequence SEQ ID NO: 9.

SEQ ID NO: 9:

The nucleotide sequence SEQ ID NO: 9 encodes a polypeptide sequence comprising the chimeric antigen sequence of the first aspect, wherein said polypeptide sequence further comprises a methionine amino acid at the N-terminus.

The term "nucleic acid", "nucleotide sequence", "polynucleotide" or "polynucleotide sequence", as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term only refers to the primary structure of the molecule. Thus, this term includes DNA of double or single strand, as well as double or single strand RNA. It also includes all types of modifications known (markers known in the art, methylation, capping, replacement of one or more of the natural nucleotides with an analogue, internucleotide modifications such as those with uncharged linkages (e.g. phosphonates of methyl, phosphorus triesters, phosphorus amidates, carbamates, etc.) and with charged bonds (for example, phosphorus thioates, phosphorus dithioates, etc.), those containing pendant moieties, such as, for example, proteins (including by e.g. nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalants (e.g. acridine, psoralen, etc.), those containing chelators (e.g. metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (for example, alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.

A third aspect of the invention relates to a plasmid, hereinafter plasmid of the present invention, comprising a nucleotide sequence according to the second aspect of the invention, wherein said plasmid comprises, preferably consists of, the sequence SEQ ID NO: 10.

SEQ ID NO: 10:

The plasmid of the present invention consists of a replication system for E. coli and a selection marker for antibiotic resistance, preferably ampicillin. In this plasmid, an efficient promoter is inserted in E. coli, such as those derived from the lactose (lac) and tryptophan (trip) operon. In front of the promoter, the gene encoding a mutant of MSP1 a is inserted that lacks six amino acids preceding the repeat peptides and the repeat peptides themselves, but contains the ten amino acids preceding the first transmembrane region of the protein beginning with a codon. ATG initiation. Finally, the Xhol and EcoRI restriction sites are inserted for the cloning of polypeptides in phase with MSP1 a for expression exposed on the E. coli membrane.

In the context of the present invention, the term "plasmid", "expression plasmid" or "expression vector", used interchangeably throughout this document, refers to a DNA fragment that has the ability to replicate in a given host and can serve as a vehicle to carry out the transcription of a sequence of interest that has been inserted into it.

A fourth aspect of the invention relates to a host cell comprising the chimeric antigen according to the first aspect, the nucleotide sequence according to the second aspect and/or the plasmid according to the third aspect.

In the context of the present invention, the term "host cell" includes any type of cell that is susceptible to transformation, transfection, transduction and the like with a nucleic acid construct or expression vector comprising a nucleotide or polynucleotide sequence encoding the fusion protein of the invention. The host cell may be eukaryotic or prokaryotic. Eukaryotic cells include, but are not limited to, yeast, insect cells, mammalian cells, plant or fungal cells. Prokaryotes include Gram-negative organisms ( for example, Escherichia coli) or Gram positive (for example, bacteria of the genus Bacillus).

In a preferred embodiment the host cell is a prokaryotic cell. In a More preferred embodiment the host cell is Escherichia coli.

A fifth aspect of the invention relates to an isolated peptide that is selected from the list consisting of peptides with sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. According to the results obtained by the inventors, these peptides make up the epitopes recognized by IgG from rabbits immunized with the complete MSP4 protein, so these peptides present immunogenic activity.

The term "isolated" refers to the peptide not being part of a larger amino acid chain, for example the chimeric antigen of the present invention or the MSP4 protein.

A sixth aspect of the invention relates to a composition, hereinafter "composition of the invention", comprising the chimeric antigen according to the first aspect, the nucleotide sequence according to the second aspect, the plasmid according with the third aspect, or at least one isolated peptide according to the fifth aspect of the invention, and at least one excipient, vehicle and/or adjuvant. In a preferred embodiment, the composition of the present invention is in liquid, semisolid form , freeze-dried, solid, tablet, capsules or pills.

In a preferred embodiment, the composition of the present invention is in liquid form and comprises the chimeric antigen according to the first aspect in a concentration of 1-1000 pg/ml; In a preferred embodiment the chimeric antigen is in a concentration of 1-500 pg/ml; In a more preferred embodiment, the chimeric antigen is at a concentration of 50-200 pg/ml; In a more preferred embodiment, the chimeric antigen is at a concentration of 100 μg/ml.

The term "excipient" refers to a substance that assists in the absorption of any of the components of the composition of the present invention, stabilizes said components or assists in the preparation of the composition. Thus, the excipients could have the function of keeping the components together, such as starches, sugars or cellulose, the function of sweetening, the function of coloring, the function of protecting the medication, such as isolating it from air and/or humidity, the function of filling a pill, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that matter that, included in the "galenic forms", is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties. of the pharmaceutical or veterinary composition and its bioavailability. Furthermore, the excipient is pharmacologically or veterinary acceptable, that is, the excipient is permitted and evaluated so that it does not cause harm to the organisms to which it is administered.

The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow better dosage and administration or give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance that is used in the medicine or pharmaceutical or veterinary composition to dilute any of its components up to a certain volume or weight; or that, even without diluting said components, it is capable of allowing better dosage and administration or giving consistency and shape to the medicine or composition. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.

The term “adjuvant” refers to a substance that increases or modulates the immune response to a vaccine. In a preferred embodiment, the adjuvant may be, without limitation, poly-iCLC, 1018 ISS, aluminum salts, Amplivax, ASI5, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC3 I, Imiquimod , ImuFact IMP321 , IS Patch, ISS, ISCOMATRIX, Juv Immune, LipoVae, F59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA. 206, Montanide ISA 50V, Montanide ISA 50 V2, Montanide ISA-51, O-432. OM-174, OM-197-MP-EC, ONTAK, PEPTEL, PLGA microparticles, resiquimod, SRL172, Virosomes, YF-17D, VEGF trap, 848, beta-glucan, Pam3Cys, and Aquifa's QS2.1. In a preferred embodiment the adjuvant is Montanide ISA 50 V2.

In each case, the form of presentation of the medication or composition will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of pharmacological or veterinary administration permitted and in a quantity therapeutically effective. Thus, the composition can be presented in a form adapted to the oral, sublingual, nasal, intramuscular, bronchial, lymphatic, rectal, intradermal, intravenous, subcutaneous or inhaled administration, but not limited to these forms.

The composition of the invention will contain a prophylactically or therapeutically effective amount of chimeric antigen according to the first aspect, the nucleotide sequence according to the second aspect, the plasmid according to the third aspect, or at least one isolated peptide according to with the fifth aspect of the invention. As used herein, the term "prophylactically or therapeutically effective amount" refers to the amount of chimeric antigen according to the first aspect, the nucleotide sequence according to the second aspect, the plasmid according to the third aspect, or at least one isolated peptide according to the fifth aspect of the invention contained in the composition that is capable of producing the desired therapeutic effect. In general, the effective amount that should be administered will depend, among other factors, on the characteristics of the subject, the severity of the disease, the method of administration, etc.

In a preferred embodiment, the composition of the present invention is a pharmaceutical or veterinary composition. In a more preferred embodiment the pharmaceutical or veterinary composition is in the form of a vaccine.

In the context of the present invention the term "vaccine" refers to an antigenic preparation used to establish the response of the immune system to a disease. They are preparations of antigens that, once inside the body, provoke the response of the immune system, through the production of antibodies, and generate immunological memory, producing permanent or temporary immunity.

A seventh aspect of the invention relates to the chimeric antigen of the first aspect, the nucleotide sequence according to the second aspect, or plasmid according to the third aspect, or at least one isolated peptide according to the fifth aspect of the invention, or the composition according to the sixth aspect for use as a medicine.

A preferred embodiment, the chimeric antigen of the first aspect refers to the nucleotide sequence according to the second aspect, or plasmid according to the third aspect, or at least one isolated peptide according to the fifth aspect of the invention, or the composition according to the sixth aspect for use in the prevention and/or treatment of infections caused by bacteria belonging to the Anaplasmataceae family in a subject.

The term “subject” refers to a human or non-human animal susceptible to infection with bacteria belonging to the Anaplasmataceae family.

In a preferred embodiment the infections are caused by bacteria belonging to the genus Anaplasma, preferably A. phagocytophilum, where the infection is anaplasmosis.

In a preferred embodiment the subject is human and the infection is human granulocytic anaplasmosis (HGA).

In another preferred embodiment the subject is an animal and the infection is anaplasmosis. In a more preferred embodiment the animal is a non-human animal, more preferably it is a non-human mammal which may be, without limitation, sheep (Ovis orientalis aries) and other ungulates, cat (Felis catus), dog (Canis lupus), rabbit (Oryctolagus cuniculus). In a more preferred embodiment the animal is sheep (Ovis orientalis aries).

In a preferred embodiment of the seventh aspect, the route of administration may be, without limitation, oral, sublingual, nasal, intramuscular, bronchial, lymphatic, rectal, intradermal, intravenous, subcutaneous or inhaled, preferably the route of administration is subcutaneous. More preferably, the route of administration is subcutaneous when the composition of the invention is in the form of a vaccine.

In the context of the present invention, "anaplasmosis" refers to the infectious disease caused by bacteria of the genus Anaplasma, where the subject suffering from said infection suffers from fever, headache, myalgia, chills and tremors, and in its most severe form can cause respiratory failure, bleeding problems, organ failure and even death. In the context of the present invention, the term "human granulocytic anaplasmosis" refers to the infectious disease caused by bacteria of the genus Anaplasma or "anaplasmosis" specifically where the affected subject is human, particularly by Anaplasma phagocytophilum, which causes fever, headache , myalgia, chills and tremors, thrombocytopenia, leukopenia and an elevation of blood transaminase. An eighth aspect of the invention refers to a method of obtaining antibodies against infections caused by bacteria belonging to the Anaplasmataceae family in a subject, preferably belonging to the genus Anaplasma, more preferably A. phagocytophilum, where said method comprises the following steps: a) Immunization of an animal with the chimeric antigen of the first aspect, the nucleotide sequence according to the second aspect, or plasmid according to the third aspect, or at least one isolated peptide according to the fifth aspect of the invention, or the composition according to the sixth aspect; b) Isolate and purify specific antibodies against bacteria belonging to the Anaplasmataceae family in a subject, preferably belonging to the genus Anaplasma, more preferably A. phagocytophilum, from a sample isolated from the animal in step a). c) Optionally humanize the antibodies of step b) for use as a medicine, preferably for the treatment and/or prevention of infections caused by bacteria belonging to the Anaplasmataceae family in a subject, preferably belonging to the genus Anaplasma, more preferably A. phagocytophilum .

The sample of step b) is a biological fluid sample. Preferably the biological fluid sample can be whole blood, serum or plasma.

The isolation and purification of step b) by a method known to a person skilled in the art such as chromatographic or affinity methods.

DESCRIPTION OF THE FIGURES

Figure 1. Production of recombinant proteins. A western blot was performed with IgG antibodies from rabbits immunized with the recombinant chimeric antigen to confirm the size of the MSP4 chimeric antigens. SEQ ID NO: 1, the antigen chimeric that contained the epitopes or regions that showed protection against A. phagocytophilum in rabbits and showed a molecular weight of 69KDa. The box indicates the position of the recombinant antigens.

Figure 2. Recognition of the chimeric protein by antibodies produced in sheep previously immunized with said protein. Samples equivalent to 10 pg of total proteins were loaded on a 12% polyacrylamide gel. For Western-blot analysis, proteins were transferred to a nitrocellulose membrane, exposed to sheep antibodies against the chimeric MSP4 protein, and revealed with anti-sheep conjugate coupled to horseradish peroxidase. The position of the recombinant chimeric protein is indicated by a box. In protein electrophoresis, Spectra (Thermo Fisher Scientific) was used as a molecular weight marker.

Figure 3. Antibody inhibition assay. Role of antibodies against chimeric recombinant proteins from immunized sheep in the inhibition of A. phagocytophilum infection of HL60 and ISE6 cells. These antibodies were compared with the protective capacity of IgGs from sheep immunized with the new chimeric MSP4 antigen object of this patent (SEQ ID NO: 1) obtained from the analysis of the MSP4 microarray. SEQ ID NO: 6 was used as a negative control. Infection levels were determined by real-time PCR of MSP4, normalizing against human β-actin for HL60 cells and against tick rpS4 for ISE6 cells. . The results were compared between the groups treated with preimmune antibodies/control and with recombinant proteins using Student's t test with unequal variance (*P < 0.05; N = 3 replicates per treatment). Peptide (+): fusion protein comprising chimeric antigen with sequence SEQ ID NO: 1. Peptide (-): fusion protein comprising the non-immunogenic peptide with sequence SEQ ID NO: 6.

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

Example 1. Microarray binding of IgG antibodies to MSP4 epitopes and data analysis. The microarray contained peptides from the MSP4 protein elongated with GSGSGSG neutral linkers at the C- and N-terminus and translated into 282 different 15-aa peptides overlapping and printed in duplicate (564 peptide spots in each copy of the array). Sera from a previous MSP4 vaccination trial (n=3) were pooled for each group and used to identify protective regions or candidate epitopes on MSP4 of that pathogen. High-resolution epitope mapping of the A. phagocytophilum MSP4 protein was performed (PEPperCHIP® Immunoassay, PEPperPRINT, Germany). The peptide microarray was mounted in an incubation tray and blocked with 1% (w/v) bovine serum albumin (BSA) in 1 X PBS 7.4 with 0.005% (v/v) Tween-20 (PBST). for 30 minutes at room temperature. After washing with PBST three times, the array was incubated with the sera overnight at 4 ^ο C. The next day, the array was washed again and incubated with a rabbit anti-lgG antibody (H+L)- Alexa Fluor 532nm (Thermo Fisher Scientific, Waltham, MA, USA) and an anti-sheep IgG (H+L)-Cy3 antibody (Sigma-Aldrich), for 45 minutes at room temperature. The array was washed, removed from the tray, and dried by centrifugation for 1 min at 2000 rpm. The resulting array was scanned with a GenePix personal 4100a microarray scanner (Molecular Devices). The median fluorescent signal intensity of each spot was extracted using MAPIX software (Molecular Devices). The protective epitopes were recognized by IgG from sera from rabbits and lambs previously vaccinated with the recombinant MSP4 protein using this method.

For data analysis, the intensity of the raw fluorescence signal was considered, which corresponded to the median of the signal intensity subtracted by the median of the background intensity of each spot and then the average was made between the duplicate spots. . Epitopes were defined as core amino acids shared by overlapping peptides with signal intensities greater than a threshold of 150 relative light units (RLU) and were used to construct two chimeric MSP4 antigens for the control of anaplasmosis, one of them containing 4 peptides. which showed protection against the pathogen in rabbits (SEQ ID NO: 1) and the other contained the non-protective region found in lambs immunized with MSP4 (SEQ ID NO: 6) and was used as a negative control.

Example 2. Production and characterization of the recombinant chimeric antigen.

The coding sequences of the new candidate protective chimeric antigens were amplified from synthetic genes optimized for codon usage in Escherichia coli (Genscript Corporation, Piscataway, NJ, USA) with sequence-specific primers, using the chimera-based membrane expression system for expression. MSP1 a, previously reported in ES2352946B1. Recombinant E. coli BL21 previously transformed with the plasmid containing the sequence encoding SEQ ID NO: 1 (plasmid with sequence SEQ ID NO: 10) was propagated in 1 L flasks containing 250 ml of Luria-Bertani broth ( LB) supplemented with 10 g of triptonel-1, 5 g of yeast extract 1-1, 10 g of NaCl 1-1, 50 μg/ml of ampicillin and 0.4% glucose (Conda SA Laboratories, Madrid, Spain ) for 2 h at 37°C and 200 rpm and then for 5.0 h after the addition of 0.5 mM final concentration of isopropyl-BD-thiogalactopyranoside (IPTG) for the induction of expression of the recombinant protein. Cell growth was monitored by measuring optical density (OD) at 600 nm. Cells were collected by centrifugation at 3900 x g for 15 minutes at 4°C, and then 1 g of cell pellet was resuspended in 5 ml of disruption buffer (100 mM Tris-HCI, pH 7.5, 150 mM NaCl, 1 mM PMSF, 5 mM MgCI2-6H2O and 0.1% (v/v) TritonX-100) and dissolved using a cell sonicator (Model MS73; Bandelin Sonopuls, Berlin, Germany). After disruption, insoluble and soluble protein fractions containing membrane-bound MSP1 a chimera antigens were collected by centrifugation at 21,500 x g for 15 minutes at 4°C and stored at -20°C until that were used for the characterization and formulations of the vaccine. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA).

For the expression and characterization of the recombinant chimeric protein, a western blot was performed. Ten micrograms of each recombinant protein were loaded on a pre-made 12% SDS-polyachlamide gel (Life Science, Hercules, CA, USA) and transferred to a nitrocellulose membrane. In protein electrophoresis, Spectra (Thermo Fisher Scientific) was used as a molecular weight marker. The membrane was blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, Ml, USA) for 2 hours at room temperature (RT) and washed three times with TBS (50 mM Tris-CI, pH 7.5, 150 mM NaCl, 0.5% Tween 20). Purified IgG from rabbits immunized with MSP1 were used at a 1:500 dilution in TBS, and the membrane was incubated overnight at 4 ^ο C and washed four times with TBS the next day. The membrane was then incubated with an anti-IgG-rabbit-peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:1,000 in TBS with 3% BSA. The membrane was washed four times with TBS and finally revealed with TMB (3,3', 5,5'-tetramethylbenzidine). stabilized substrate for HRP (Promega, Madrid, Spain) according to the manufacturer's recommendations.

The molecular weight of the chimeric protein fused to MSP1a was estimated to be between 65 and 70 kDa by SDS-PAGE (Fig. 1). This value was in accordance with the theoretical estimate of 69 kDa, of which 12 kDa correspond to the chimeric antigen object of the invention and the remaining kDa correspond to MSP1 a.

Example 3. Vaccine formulation and immunization of sheep.

For the vaccine formulation, the recombinant proteins were adjuvanted in Montanide ISA 50 V2 (Seppic, Paris, France), up to a final protein concentration of 100 μg/ml.

On the one hand, a vaccine was formulated with the fusion protein with the sequence SEQ ID NO: 8 which corresponds to a polypeptide or fusion protein that comprises the chimeric antigen made up of the antigenic fragments or peptides of MSP4 (SEQ ID NO: 1), fused to the MSP1 a protein fragment (SEQ ID NO: 7).

On the other hand, a vaccine was formulated with a fusion protein with an amino acid sequence SEQ ID NO: 1 1, which corresponds to a polypeptide or fusion protein that comprises the chimeric antigen that comprises the peptide with sequence SEQ ID NO: 6, fused to the MSP1 a protein fragment (SEQ ID NO: 7).

SEQ ID NO: 1 1 :

The amino acid sequence SEQ ID NO: 11 corresponds to a fusion protein that comprises a non-immunogenic MSP4 peptide fused, through the N-terminal end, to the MSP1 a fragment (sequence SEQ ID NO: 6 + SEQ ID NO: 7).

So that the chimeric proteins of both vaccine formulations comprised the sequence SEQ ID NO: 7, differing in the sequence SEQ ID NO: 1 and SEQ ID NO: 6.

Two groups of 3 sheep with a similar live weight were formed, one of the groups was immunized with the protective chimeric antigen that contained the epitopes recognized by the IgG of the rabbits immunized with MSP4 (SEQ ID NO: 1) the other group of the sheep was immunized with the peptide recognized by the non-protective IgG of lambs previously immunized with MSP4 (SEQ ID NO: 6). The sheep in each group were injected subcutaneously with 100 pg of the antigens into the loose skin of the axilla (armpit) using a sterile syringe with a removable 20G x 1" (9.0 x 25mm) needle and taking aseptic precautions. Sheep were immunized three times on days 0, 20 and 55 and blood samples were taken from the jugular vein of each sheep before each immunization.

Sera from sheep immunized with the recombinant chimeric protein with the sequence SEQ ID NO:1 recognized the fused protein (MSP4 chimeric antigen-MSP1 fragment a) by Western-blot (Fig. 2). These results indicated that the protective epitopes identified in rabbit maintained their antigenicity in sheep immunized with the chimeric protein.

Example 4. Antibody inhibition assay.

Samples were prepared by culturing the Ixodes scapularis tick cell line ISE6 in L15B300 medium and human promyelocytic HL-60 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine and 25 mM Hepes buffer at 36.5C with 5% CO2 (3 replicates each). The determination of the inhibitory effect of IgG antibodies from immunized lambs and rabbits (the IgGs come from the study previous Contreras M., et al. 2017. Front. Cell. Infect. Microbiol. 7:307) and from sheep immunized with the protective chimeric antigen and the negative control peptide 15 days after the third immunization was carried out using IgG purified from the sera using the NAb Protein G centrifugation kit (Thermo Fisher Scientific , Waltham, MA, USA) following the manufacturer's recommendations. Sheep IgG antibodies from preimmunized sera were also used as a control. ISE6 cells were pooled and used to seed 24-well plates with each well receiving 1 x 106 cells 48 h before inoculation with human A. phagocytophilum isolate NY18. Purified rabbit or sheep IgG (2 mg/ml) were mixed with the inoculum (1:1) for 60 min before being placed on the cell monolayers. Each monolayer then received 100 μl of the inoculum plus the IgG mixture and the plates were incubated at 34°C for 30 min. The inoculum was removed from the wells and the cell monolayers were washed three times with PBS. Complete medium (1 ml) was added to each well and the plates were incubated at 34°C. The control of each assay included an inoculum incubated with rabbit or sheep preimmune IgG. After 7 days, cells were recovered from all wells and frozen at −80°C until detection of A. phagocytophilum by PCR after DNA extraction with TriReagent (Sigma-Aldrich) according to the manufacturer's recommendations. A. phagocytophilum infection levels were determined by real-time PCR of major surface protein 4 (msp4), as previously described (Contreras M., et al. 2017. Front. Cell. Infect. Microbiol. 7:307). The results were compared between treatments using the student's t test with unequal variance (P = 0.05; N = 3 biological replicates).

While rabbit IgG antibodies against the recombinant MSP4 protein of A. phagocytophilum inhibited pathogen infection of HL60 and ISE6 cells as in previous studies, IgG from sheep immunized with the chimeric protective peptide object of this invention (SEQ ID NO: 1, peptide (+)) affect infection by the pathogen, but IgG from sheep immunized with the peptide SEQ ID NO: 6 (peptide (-)) and IgG from lambs immunized with MSP4 from the previous study did not protect against A. phagocytophilum infection (Fig. 3).

These results showed differences in the IgG response between immunized rabbits and lambs and provided support that the design of the new protective chimeric antigen improves the protective capacity of the MSP4 antigen.

Claims

1. A chimeric protective antigen against A. phagocytophilum comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 1.

2. Chimeric antigen according to claim 1 characterized in that it is linked by one of its N or C terminal ends to an amino acid sequence of a bacterial membrane anchoring protein or protein fragment; preferably where said amino acid sequence has an identity of at least 80%, 90%, 95% or 98% with the amino acid sequence SEQ ID NO: 7, more preferably where said amino acid sequence comprises, even more preferably consists of , SEQ ID NO: 7.

3. Chimeric antigen according to claim 2 comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 8.

4. A nucleotide sequence encoding the chimeric antigen according to any of claims 1 to 3.

5. Nucleotide sequence according to claim 4 comprising the nucleotide sequence SEQ ID NO: 9.

6. A plasmid comprising a nucleotide sequence according to any of claims 4 or 5.

7. Plasmid according to claim 6 comprising the sequence SEQ ID NO: 10.

8. A host cell comprising the chimeric antigen according to any of claims 1 to 3, the nucleotide sequence according to any of claims 4 or 5, and/or the plasmid according to any of claims 6 or 7.

9. Host cell according to claim 8 which is a prokaryotic cell, preferably Escherichia coli.

10. Isolated peptide that is selected from the list consisting of the peptides with sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. A composition comprising the chimeric antigen according to any of claims 1 to 3, the nucleotide sequence according to any of claims 4 or 5, the plasmid according to any of claims 6 or 7, or at least one peptide according to claim 10, and at least one excipient, vehicle and/or adjuvant. Composition according to claim 11 characterized in that it is a pharmaceutical or veterinary composition, preferably in the form of a vaccine. Chimeric antigen according to any of claims 1 to 3, nucleotide sequence according to any of claims 4 or 5, plasmid according to any of claims 6 or 7, peptide according to claim 10, or composition of according to any of claims 1 1 or 12 for use as a medicine. Chimeric antigen according to any of claims 1 to 3, nucleotide sequence according to any of claims 4 or 5, plasmid according to any of claims 6 or 7, peptide according to claim 10, or composition of according to any of claims 1, 1 or 12 for use in the prevention and/or treatment of infections caused by bacteria belonging to the Anaplasmataceae family in a subject. Chimeric antigen, nucleotide sequence, plasmid, peptide or composition for use according to claim 14 wherein the infection is caused by bacteria of the genus Anaplasma, preferably A. phagocytophilum. Chimeric antigen, nucleotide sequence, plasmid, peptide or composition for use according to any of claims 14 or 15 where the subject is human. Chimeric antigen, nucleotide sequence, plasmid, peptide or composition for use according to claim 16 wherein the infection is human granulocytic anaplasmosis (HGA). Chimeric antigen, nucleotide sequence, plasmid, peptide or composition for use according to any of claims 14 or 15 wherein the subject is a non-human animal, preferably a non-human mammal, and the infection is anaplasmosis.

19. Chimeric antigen, nucleotide sequence, plasmid, peptide or composition for use according to any of claims 13 to 18, wherein the route of administration is subcutaneous.