EP1576003A1 - Peptides pour la prevention, le diagnostic et le traitement de la leptospirose animale et/ou humaine - Google Patents
Peptides pour la prevention, le diagnostic et le traitement de la leptospirose animale et/ou humaineInfo
- Publication number
- EP1576003A1 EP1576003A1 EP03780234A EP03780234A EP1576003A1 EP 1576003 A1 EP1576003 A1 EP 1576003A1 EP 03780234 A EP03780234 A EP 03780234A EP 03780234 A EP03780234 A EP 03780234A EP 1576003 A1 EP1576003 A1 EP 1576003A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- protein
- test
- seq
- leptospires
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Definitions
- the subject of the present invention is peptides capable of inducing protection against leptospirosis (in particular by vaccine), immunogenic compositions (diagnostic and / or therapeutic reagents), comprising one or more of these peptides or the antibodies, nucleic acids or vector derived from it.
- immunogenic compositions diagnostic and / or therapeutic reagents
- These compositions are particularly advantageous since they make it possible to induce an immune response or protection against different pathogenic strains, in particular against several pathogenic serogroups different from leptospires and to ensure a diagnosis common to all clinical cases whatever the species affected. .
- Pathogenic leptospires are responsible for infectious diseases affecting humans and animals.
- Leptospirosis is one of the zoonoses monitored by the WHO due to its global distribution and the severity of the disease (lethal in 2 to 20% of patients depending on the earliness of the diagnosis and treatment).
- the economic impact is significant, the disease being responsible for reproductive disorders in farm animals (ruminants or pigs) or for serious clinical signs altering the health of the companion animal that is dog, or that of the sport animal that is the horse.
- leptospirosis is illustrated in particular by two aspects: firstly their impact on public health (hospitalizations, incapacity for work, sometimes death, implementation of vaccinations as part of protection against occupational diseases in certain countries including France), on the other hand their economic impact (cost of implementing public health programs, loss of production for farm animals and cost of vaccines used in many countries to fight against this drop in production, emotional loss of the pet or / and cost of vaccines currently available in dogs in almost all countries of the world, and further financial loss due to the deterioration of horse sporting performance).
- the fight against these infections is particularly difficult, given the bacteriological complexity of these germs and the diversity of reservoirs made up by wild animals, carriers, excretors and disseminators of these germs through their urine.
- the bacteriological complexity of these bacteria is such that two classifications of these germs currently coexist: a serological classification and a genomic classification.
- Leptospira interrogans sl have been classified in the same pathogenic species Leptospira interrogans sl as opposed to aquaculture saprophytic strains and classified in Leptospira biflexa sl
- the Leptospira interrogans species is divided into more than 25 different serogroups, each of these serogroups being itself composed of several serovars having between them sufficient antigenic communities for their grouping. There are thus some 220 different serovars for the pathogenic species. This classification is made on the basis of antigens inducing agglutinating antibodies, this is why it is still the only one used within the framework of the serological diagnosis of the infection.
- Leptospira interrogans if has been split into different species called genomospecies: Leptospira interrogans sensu stricto (ss), Leptospira kirshneri, L. borgpetersenii, L. wellii, L. noguchii, L. santarosai, L. meyeri, L.fainei ....
- leptospirosis The clinical expression of leptospirosis is very polymorphic depending on the infecting strains but also on the species. Thus, humans, sensitive to leptospirosis, generally develop acute forms of rapid evolution, with hepatic, renal or pulmonary involvement. Early diagnosis generally conditions the effectiveness of antibiotic treatment.
- the French vaccine directed against Icterohaemorrhagiae provides protection only against this serogroup considered to be the most pathogenic.
- vaccination against the two serogroups considered to be dominant in this species Icterohaemorrhagiae and Canicola
- the vaccine only protects against these two serogroups and does not prevent infection with a wild strain belonging to another serogroup. This explains the development of chronic disorders, still poorly identified by veterinary practitioners judging that a dog vaccinated against "leptospirosis cannot develop a chronic form linked to an infection by a wild strain.
- any suspicion of leptospirosis is generally the subject of experimental confirmation methods .
- Two parts of the diagnosis (and screening) may therefore be necessary, depending on the duration of the disease's progression.
- direct identification of the germ is essential.
- the indirect demonstration by the serological response is generally sufficient in terms of diagnosis, but, in a second step, the recognition of animals carrying germs, and therefore excretors, requires the demonstration of the germ .
- the direct detection of the pathogen of the germ can be carried out essentially by two methods: isolation and PCR.
- Bacteriological isolation is a very cumbersome method.
- the natural fragility of leptospires means that only samples treated within a limited time allow the possible isolation of a strain of leptospires.
- the probability of isolating a leptospire is generally low, given the requirements of these germs; in addition, the response time can be up to several weeks, given the slow development of the crops.
- PCR has therefore proven to be a much more interesting tool in terms of diagnosis.
- this method is currently only developed in humans, for which it is implemented from a blood sample from the patient in the acute phase. Carrying out PCR as a diagnostic method on tissues such as abortion products is still only at the experimental stage.
- a method which would be useful in breeding it is not currently operational.
- the serological diagnosis of the germ consisting of detecting antibodies indicating an infection, is generally carried out by the Microagglutination test (MAT) which is currently the reference method.
- MAT Microagglutination test
- the disadvantages of this method are linked to the bacteriological heterogeneity of leptospires.
- the method requires the use of live germs (the culture of which is delicate) and, moreover, is based on the detection of agglutinating antibodies induced by the surface antigens of leptospires, antigens which have served for their classification and therefore express l heterogeneity of these bacteria.
- This means that the implementation of serological diagnosis requires that the patient's serum be brought into contact with a living strain representative of each of the different serogroups, which means that the number of manipulations for the same serum must be increased.
- the vaccines marketed are composed of a suspension or more exactly of an addition of suspensions of bacteria representative of each of the serogroups necessary for the protection of the species of destination of the vaccine. Given the great diversity of serogroups, a choice is required depending on the species and the epidemiological conditions. Thus, the usual vaccines used in dogs combine a suspension of bacteria inactivated from the Icterohaemorrhagiae serogroup and one from the Canicola serogroup.
- Vaccines used in pigs and ruminants in the USA combine suspensions of serovars belonging to serogroups: Icterohaemorrhagiae, Pomona, Grippotyphosa, Canicola and Sejro ⁇ (serovar hardjo).
- the vaccine for human use marketed in France at present comprises the Icterohaemorrhagiae serogroup, etc.
- the problem with these vaccine preparations is their failure in terms of total protection against leptospirosis.
- a vaccinated individual whose vaccination is maintained in the manner prescribed by the vaccine producers is protected against infection and / or disease induced by a wild strain belonging to the only serogroups present in the vaccine. It is therefore only protected (and still this protection may be imperfect) only against infection by one or more given serogroups, represented in the vaccine preparation.
- a first step it was demonstrated that the presence of whole bacterial bodies was not essential for the induction of homologous protection.
- response or homologous protection is meant the protection induced by a preparation composed by leptospires belonging to the same serogroup as the leptospires used in the virulent test carried out on sensitive laboratory animal and making it possible to assess the effectiveness and the authenticity of the protection conferred. This has been demonstrated by immunizing animals with a total extract of leptospires obtained by the bursting of bacteria.
- a second step it has been demonstrated that total extracts from different serogroups make it possible to induce heterologous protection within the species L. interrogans si.
- the fourth step consisted in identifying the antigens differentiating the pathogenic species from the saprophytic species: For this, comparisons of electrophoretic or immunotransfer profiles of total extracts of leptospires from different pathogenic or non-pathogenic serogroups were carried out and show antigenic bands ranging from 14 to 200 kD, of which 24 are common to the pathogenic species and in particular the zone 21-26 kD which is serogroup-specific (Gitton X, André-Fontaine G, André F, Gagro JP. "Immunoblotting study of the antigenic relationships among eight serogroups of Leptospira.
- LPS corresponds to the external antigenic structures responsible for the production of agglutinating antibodies used as the basis for serological classification, but also responsible for the protective effect of vaccine preparations. According to the prior art, it was therefore necessary for a vaccine to be capable of inducing the production of agglutinating antibodies at significant levels for protect an animal from infection with a homologous leptospire.
- the sixth step consisted in checking the capacity of a protein extract of an Autumnalis or Canicola strain purified of any trace of LPS to induce homologous protection in the same way as purified LPS but also to induce protection against a test virulent heterologous observed in previous tests with total extracts.
- an eighth step degenerate nucleotide probes were constructed, the PPL gene was cloned, and the production of recombinant PPL protein was carried out and allowed the production of monoclonal antibodies.
- the PPL gene has been inserted into a non-replicating human adenovirus and effective protection against virulent tests carried out on the gerbil has been observed after administration of these recombinant viruses, in particular as vaccines (Branger C, Sonrier C, Chatrenet B, Klonjkowski B , Ruvoen-Clouet N, Aubert A, Andre-Fontaine G, Eloit M. "Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.” Infect Immun. 2001 Nov; 69 ( 11): 6831-8.).
- the recombinant protein produced by a prokaryotic system does not provide significant protection against the different strains of pathogenic leptospires.
- WO 99/42478 describes a membrane protein LipL32 which is present in certain pathogenic strains of leptospire. This protein contains 272 amino acids. It can be used to induce an immune response against certain strains of leptospires.
- WO 96/36355 describes two membrane proteins of pathogenic strains of leptospire, LipL1 and LipL2, with a weight of 35 kDa and 41 kDa respectively. These proteins are used to produce an immune response against certain strains of leptospires. However, it is by no means envisaged in this document that this protein makes it possible to induce heterologous protection against different strains of leptospires.
- the inventors have therefore set themselves the objective of identifying peptides capable of inducing effective protection against the various strains of pathogenic lepstopires, this protection being able to be induced directly, without passing through the expression of these peptides in an adenovirus.
- the inventors have also set themselves the objective of developing a method for the early detection of infection by the different strains of pathogenic leptospires in order to allow a diagnosis of leptospirosis at a stage of infection where it is effective to administer treatment to the individual.
- the present invention now makes it possible to solve the drawbacks of the various methods of vaccination, screening, diagnosis and treatment, described in the prior art.
- the present invention indeed describes the identification of an antigenic motif of leptospire: the PP peptide, common to multiple serogroups of leptospires, in particular pathogenic leptospires.
- the present invention also allows the production of polypeptides, peptides and antibodies, usable either in vaccine approaches or in effective therapeutic approaches and capable of inducing protection against multiple serovars of pathogenic leptospires.
- the present invention thus makes it possible to carry out tests for the detection or screening of pathogenic leptospires themselves or of the infection which they cause, not restricted to particular serovars.
- the present invention also makes it possible to carry out simpler detection tests than the MAT methods described in the prior art, since they do not require the culture of germs.
- the current the invention furthermore describes nucleic acids, vectors, probes, primers and also recombinant cells which can be used for the production of polypeptides, peptides or proteins or for the detection of leptospires or their products in any test, biological sample (for example biological fluids such as blood, plasma, urine, milk, cerebrospinal fluid, tissue, organ, cell culture, etc.) or other origin (such as a sample contaminated with biological materials, such as river water, standing water , drinking water, stored water used by companies such as slate quarries, market gardeners, etc.
- biological sample for example biological fluids such as blood, plasma, urine, milk, cerebrospinal fluid, tissue, organ, cell culture, etc.
- other origin such as a sample contaminated with biological materials, such as river water, standing water , drinking water, stored water used by companies such as slate quarries
- the first object of the invention is a peptide called PP, fragment of a PPL protein of 32 kDa described in International Application WO01 / 59123.
- This PP peptide consists of 25 amino acids and is represented by the sequence SEQ ID No: 1 shown below:
- sequence SEQ ID NO: 1 and, more generally, the compounds of the present invention have unpredictable advantages compared to proteins of the prior art: - obtaining protection against the different strains of pathogenic leptospires to from a single compound,
- the invention also relates to homologs of the PP peptide, its pharmaceutically acceptable salts, its functional fragments, its chemical analogs and certain chemical derivatives of this peptide.
- homologs is meant within the meaning of the present invention peptides whose amino acid sequence has at least 60% similarity to the sequence SEQ ID NO: 1, even more preferably 70%, even more preferably still 80%, of preferably at least 90%, and even more favorably at least 95%), or better, 98% similarity with the sequence SEQ ID NO: 1.
- X% between a peptide P and the sequence SEQ ID NO: 1 it is meant that: when the sequence of P is aligned opposite SEQ ID NO: 1, in the same direction, X%> of the amino acids of P are identical to the corresponding amino acid of SEQ ID NO: 1 or are replaced by an amino acid of the same class, it being understood that if the sequences are not of the same length, a space may optionally be placed between the amino acids of the sequence concerned.
- the degree of homology can be assessed by methods well known to those skilled in the art (e.g., WILBUR WJ et al Proceedings of the National Academy of Sciences USA 80, 726-730 (1983); MYERS et al, Comput. Appl Biosci. 4, 11-17 (1988)).
- the homology with SEQ ID NO: 1 extends to peptides comprising from 15 to 40 amino acids and whose sequence, once aligned with SEQ ID NO: 1, with the spaces placed between the acids suitable amines, has a similarity included in the values indicated above.
- the homology extends to peptides comprising from 20 to 30 amino acids, even more preferably from 22 to 28 amino acids.
- amino acids of the same class is meant an amino acid having substantially identical chemical properties.
- this term is understood to mean amino acids having substantially the same charge, and / or the same hydrophilicity or hydrophobia, and / or the same aromaticity.
- Such amino acid groupings generally include:
- amino acid substitutions can be envisaged within the meaning of the present invention in which an amino acid is replaced by another amino acid of the same class, that is to say having comparable properties, the amino acid of substitution being an unnatural amino acid.
- This is the case, for example, of the enantiomers and diastereoisomers of natural amino acids, hydroxyproline, norleucine, ornithine, citrulin, cyclohexylalanine, ⁇ -amino acids such as 3-aminopropionic acid, ⁇ -amino acids such as 4-amino-butyric acid.
- Such amino acids are well known to those skilled in the art and can be prepared by known methods.
- substitution of one or more amino acids with an unnatural amino acid carrying a group allowing the detection of the peptide, such as an amino acid carrying a fluorescent, photoactivable group or a radiolabelled amino acid.
- a particular example of homolog is constituted by the functional fragments of the peptides according to the invention: these are peptides corresponding to one of the sequences according to the invention in which one or more amino acids are removed from the sequence but which retain a immunogenic activity comparable to that described for the PP peptide, in particular a comparable antibody titer.
- the preferred functional fragments of the peptides according to the invention are those for which at most six amino acids, more preferably 4 amino acids, more preferably still 2 amino acids, are removed. Even more preferably, those for which an amino acid is removed.
- substitutions of amino acids of the same class allow the peptide to retain its immunogenic activity.
- Appropriate substitutions can be determined by testing the antimicrobial properties of the peptides obtained using activity tests such as those described below.
- the invention relates more specifically to molecules inducing an antibody titer and / or conferring protection comparable to those capable of being obtained by the use of the PP peptide.
- the term salts relates both to the amine salts of a carboxyl function of the peptide chain as well as to the acid addition salts with an amino group of this same polypeptide chain.
- the salts of a carboxyl function can be formed with an inorganic or organic base.
- Inorganic salts include, for example, alkali metal salts such as sodium, potassium and lithium salts; alkaline earth salts such as for example calcium, barium and magnesium salts; ammonium salts, ferrous, ferric salts, zinc, manganese, aluminum, magnesium salts.
- the salts with organic amines include those formed for example with trimethylamine, triethylamine, tri (n-propy ⁇ ) amine, dicyclohexylamine, triethanolamine, arginine, lysine, histidine, ethylenediamine, glucosamine , methylglucamine, purines, piperazines, piperidines, caffeine, procaine.
- Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid; salts with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid , malic acid, ascorbic acid, benzoic acid.
- mineral acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid
- organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid , malic acid, ascorbic acid, benzoic acid.
- Chemical derivatives of the peptides according to the invention include the alpha-amino peptide compounds, the N-alpha acyl substituted derivatives of the RCO- form, in which R represents a linear, branched alkyl, alkenyl, alkynyl, aryl or aralkyl group , or cyclic comprising from 1 to 50, preferably from 1 to 8 carbon atoms.
- the preferred N-alpha acyl group is the acetyl group.
- Such amine-terminal substituents can increase the activity of the peptide by slowing down or preventing the enzymatic degradation of the peptides in vivo.
- Other chemical derivatives of the peptides according to the invention include derivatives substituted on the C-terminal acid function by a group chosen from - NH 2 , alkyloxy, alkylthio or alkylamino of the form -OR, -SR or -NHR, in which R can represent an alkyl, alkenyl, alkynyl, aryl chain or an aralkyl group, linear, branched or cyclic comprising from 1 to 50, preferably from 8 to 50 carbon atoms.
- Another kind of chemical derivative of the peptides according to the invention includes derivatives carrying a pharmacophore substituent, such as a fluorescent group, a photoactivable group, a radiolabelled group or any other group allowing detection by spectroscopy and quantitative evaluation. of the peptide according to the invention in a biological sample without degradation of the biological sample.
- the amino acids of which the peptides are made up are L enantiomers.
- one or more amino acids of the peptide sequence can be replaced by its D enantiomer.
- one or more peptide amide bonds can be replaced by an isostere bond such as: -CH 2 NH-, CH 2 S-, -CH 2 CH 2 -, -CH ⁇ CH- (cis and trans), - COCH 2 -, -CH (OH) CH 2 - and -CH 2 SO-.
- Derivatives of the peptides according to the invention include polymers of these peptides, more preferably dimers of these peptides via, for example, a thiol group of cysteine or oligomers by grafting peptides onto polylysines (MAP), or coupling of these peptides to carrier proteins (BSA,
- MAP polylysines
- BSA carrier proteins
- the peptides which are the subject of the present invention can be easily obtained by any means known to a person skilled in the art, in particular by synthesis.
- a subject of the invention is also peptides comprising a sequence SEQ ID NO: 1 or peptides comprising a functional fragment of these peptides, a homolog, a chemical analog of these peptides or a chemical derivative of these peptides.
- the terminal amino acids of the PP peptide comprise, for one, a free terminal amino function, and for the other, a free terminal carboxyl function, these two functions each being capable of being engaged in a peptide bond with C - terminal acid, respectively the N - terminal amine of another peptide fragment.
- these peptides contain at most 100 amino acids, preferably at most 80 amino acids, even more preferably at most 60 amino acids and advantageously at most 40 amino acids.
- the immunogenic character of the polypeptides or peptides of the invention can be verified in various ways known to a person skilled in the art.
- polypeptides can be brought into contact with antibodies (or serum of infected subjects), the detection of antigen-antibody complexes indicating the capacity of the polypeptides of the invention to carry epitopes or immunogenic fragments.
- proteins comprising a peptide as defined above, coupled with a carrier protein, such as for example the KLH protein or the BSA protein. This coupling makes it possible to increase the immunogenicity of the peptide of the invention.
- carrier proteins are illustrated in documents US-4,608,251; US-5,945,104; WO 90/15878.
- the coupling of the peptide of the invention and the carrier protein can be done in a known manner by chemical coupling or by addition of the DNA sequence of the peptide before or after that of the carrier protein in a plasmid then expression and purification of the protein that it is designated by the name of fusion protein.
- Another subject of the invention relates to antibodies directed specifically against a protein, a polypeptide or a peptide according to the invention. They are preferably anti-leptospire antibodies, binding an epitope present in a protein, a polypeptide or a peptide according to the invention.
- the antibodies of the invention are preferably specific for leptospires, in particular pathogenic, although that a weaker (or non-specific) binding can be observed experimentally with other antigens.
- the antibodies according to the invention can be polyclonal or monoclonal antibodies. They are generally produced by immunizing an animal with a peptide, a polypeptide or a protein, according to the invention and recovering the serum, milk or the egg if they are birds (to obtain the antibodies polyclonal) or thymus or spleen cells, for the production of hybridomas producing monoclonal antibodies.
- the antibodies according to the invention are more preferably antibodies recognizing the PP peptide of sequence SEQ ID NO: 1 as defined above, a functional fragment thereof, a homolog, a chemical analog or a chemical derivative thereof. this.
- the antibodies of the invention advantageously have the capacity to recognize at least two pathogenic strains of leptospire belonging to different serogroups, more preferably at least 3 pathogenic strains of leptospire, in particular belonging to different genomic species.
- the antibodies according to the invention are polyclonal antibodies prepared by immunization of an animal with the PP peptide.
- the animal can be a rodent (rat, mouse, gerbil, hamster, guinea pig ...), a primate, a rabbit, a pig, a horse, a cattle, a bird, etc.
- Polyclonal antibodies are generally recovered from the serum of immunized animals or eggs, according to protocols known to those skilled in the art.
- the antibodies according to the invention are monoclonal antibodies prepared from hybridomas obtained by fusion between an immortalized cell (for example a myeloma) and an antibody-producing cell taken from an animal immunized with the PP peptide as defined above.
- the animal can be a rodent (rat, mouse, gerbil, hamster, guinea pig ...), a primate, a rabbit, a pig, a horse, a cattle, a bird, etc.
- the antibodies of the invention in particular the monoclonal antibodies, can also be humanized, that is to say modified artificially to contain regions of heavy or light chains of human origin.
- the invention also relates to fragments or derivatives of such antibodies, for example Fab, F (ab ') 2 fragments, ScFv (single chain antibody), etc.
- the antibodies of the invention can be used for example for the detection of pathogenic strains of leptospires or of products secreted by these same leptospires in test samples (in particular blood and milk samples , cerebrospinal fluid or urine), or to induce (emergency) protection against leptospire infections.
- nucleic acids may be DNAs or RNAs, in particular recombinant, genomic, synthetic or semi-synthetic DNAs or also mRNAs, or fragments or derivatives thereof.
- the nucleic acids can be obtained from banks, clones from leptospire bacteria (in particular by PCR), produced by artificial synthesis using synthesizers of nucleic acids, or also prepared using combinations of these methods (enzymatic digestions, ligatures, cloning, modifications, etc.).
- the nucleic acids can also be modified to improve the use of codons, eliminate cryptic promoters, reduce secondary structures, etc.
- the invention also relates to vectors comprising a nucleic acid as defined above. It can be a vector of plasmid, cosmid, episome, artificial chromosome, phage, virus, etc. type. It is more preferably a plasmid, for example a replicative or integrative plasmid in bacteria or eukaryotic cells (yeasts, mammalian, bird, insect cells, etc.) or a recombinant virus , in particular defective (such as a poxvirus, an adenovirus, a retrovirus, a baculovirus, a herpesvirus, etc.).
- a plasmid for example a replicative or integrative plasmid in bacteria or eukaryotic cells (yeasts, mammalian, bird, insect cells, etc.) or a recombinant virus , in particular defective (such as a poxvirus, an adenovirus, a retrovirus, a baculovirus, a her
- Plasmid vectors can be prepared by conventional techniques using commercially available plasmids, such as pUC, pBR, pCN, etc.
- the invention also relates to any recombinant cell comprising a nucleic acid or a vector as defined above.
- the recombinant cell can be a bacterium (for example an E. coli strain), or a eukaryotic cell, in particular a yeast, a mammalian, bird, or insect cell, etc.
- the recombinant cells can be used in particular for the production of the polypeptides of the invention, as well as as models for the search for compounds capable of neutralizing or antagonizing the activity of the PP peptide.
- the invention further relates to any non-human transgenic organism, in particular any non-human mammal, any bird, or any plant organism comprising a nucleic acid as defined above in its cells.
- these transgenic organisms are obtained by homologous recombination.
- mammals rodents, canines, rabbits, goats, pigs, etc.
- plants can be used in particular for the production of the polypeptides of the invention and for the identification of compounds for therapeutic or vaccine purposes, for example.
- nucleotide probes and / or primers which can be used for the detection and / or amplification of leptospire nucleic acids, and in particular for the detection of the presence of a pathogenic leptospire strain or of product secreted by it in a test sample (in particular a biological product or contaminated by a biological product).
- the nucleotide probes according to the invention advantageously comprise a nucleic acid as defined above. They are preferably single-stranded, and can be labeled, for example by radioactive, enzymatic, fluorescent, chemical labeling, etc.
- a probe according to the invention preferably comprises a sequence complementary to all or part of the nucleotide sequence SEQ ID NO: 1 or to one of the peptides according to the invention as defined above.
- the probes of the invention can be used to detect the presence of a pathogenic strain of leptospire or of product secreted by it in any sample, in particular a biological sample.
- a preferred probe according to the invention comprises the complete sequence of the nucleotide sequence coding for PP, or a fragment thereof.
- the nucleotide primers according to the invention are oligonucleotides, of a size generally less than 40 bases, comprising the sequence of a nucleic acid as defined above.
- the primers can be used for the amplification of Leptospires nucleic acids, in particular for the amplification of the sequence coding PP or a part thereof, for example by PCR reaction.
- the invention relates more particularly to any pair of primers allowing the amplification of a region of the sequence coding PP as defined above.
- the amplified region comprises at least 50 bp.
- the probes, primers or oligonucleotides of the invention are preferably complementary to at least one region of the sequence coding PP.
- the complementarity is generally perfect, so as to ensure better selectivity of hybridization. However, some mismatches can be tolerated.
- These probes or oligonucleotides can be synthesized by any technique known to those skilled in the art, for example by cleavage from the nucleic acids described above, or by artificial synthesis, or by combining these techniques.
- the probes and primers are particularly useful for the detection of the presence of pathogenic strains of leptospires, and / or the diagnosis of leptospiroses, as will be detailed below.
- the detection of antibodies directed against an antigen specific for pathogenic strains is therefore particularly advantageous.
- the present invention can thus be used on the diagnostic, vaccine, therapeutic and / or experimental level.
- the PP peptide of the invention can be used as an antigenic motif capable of demonstrating antibodies produced during infection by a pathogenic strain, whatever its serogroup of membership. This therefore allows the experimental diagnosis of leptospirosis to be ensured by many laboratories with ordinary equipment (example ELISA or dot) while MAT requires maintenance of live cultures and its standardization, therefore, is very difficult.
- a particular object of the invention therefore resides in the use of a polypeptide, peptide or a protein as defined above for detecting in vitro the presence of anti-leptospire antibodies in a biological test sample (in particular a biological sample such as blood, serum, milk, urine, tissue, etc.
- Another object of the invention lies in an in vitro method for detecting the presence of anti-leptospire antibodies in a test sample, this method comprising bringing this sample (or a dilution) into contact with a polypeptide, peptide or a protein as defined above, and the demonstration of the formation of antigen-antibody complexes.
- the antibodies (or fragments or derivatives of antibodies) according to the invention allow the direct detection of the pathogenic agent or of secreted products present in a test sample (such as a biological sample or another type of sample such as water).
- a test sample such as a biological sample or another type of sample such as water.
- the demonstration can be carried out by any immunological method known to a person skilled in the art, such as by ELISA capture, RIA, direct or sandwich tests, etc., or by immunological revelation of this peptide in pathological samples or not.
- Another object of the invention therefore lies in the use of an antibody (or fragment or derivatives of antibodies) according to the invention, in particular of one or several monoclonal antibodies of the invention, for the detection of the presence of a pathogenic strain of leptospire or of its secreted products in a sample, in particular biological.
- Another object of the invention resides in a method for detecting the presence of a pathogenic strain of leptospire or of its secreted products in a sample, comprising bringing this sample into contact (or a dilution, or a concentration) with an antibody (or fragment or derivatives of antibodies) according to the invention, and the demonstration of the formation of antigen-antibody complexes.
- polypeptides, peptides, proteins and antibodies can be used in soluble form, or immobilized on solid or semi-solid supports, of the filter, silica, glass, plate, bead, etc. type.
- immobilized form advantageously makes it possible to simplify the detection or the experimental diagnosis of leptospirosis.
- These compounds can also be labeled, for example by means of fluorescent, enzymatic, biological or radioactive labels.
- revelation of antigen-antibody complexes can also be carried out using an additional labeled antibody, or according to known immunological techniques (ELISA, RIA, sandwich, capture, etc.).
- Another detection (or screening) method lies in bringing a test sample into contact with a nucleotide probe of the invention, and demonstrating hybridization between said probe and said sample. More preferably, the test sample is treated beforehand so as to make the nucleic acids which it contains accessible to a hybridization reaction.
- the treatment may consist in breaking the cell membranes, for example by chemical treatment (detergent) and / or mechanical treatment (ultrasound, freeze-thaw, etc.). To this treatment can be added methods of concentration (filtration, centrifugation, etc.) of the samples, in particular non-biological samples such as water samples.
- the sample, in particular biological, thus treated is subjected to an amplification reaction, using primers of the invention, prior to the hybridization reaction with the probe.
- Amplification can be carried out under conventional conditions.
- Hybridization can be carried out on supports, on which the probe is immobilized (filters, glass, silica, etc.).
- the stringency conditions of the hybridization can be adjusted by a person skilled in the art, by adapting the temperature and / or the salinity of the media.
- the invention further relates to a kit for carrying out the methods of the invention, comprising a probe or an oligonucleotide or a pair of primers as described above.
- the kits of the invention advantageously comprise the reagents suitable for an amplification reaction and / or hybridization, and possibly a support for such reactions (filters, membranes, chips, etc.).
- the present invention can also be used therapeutically and preventively. Indeed, although the pathogenic mode of action of the PP peptide is not yet defined, the present application shows that it is capable of inducing protection.
- the administration of antibodies directed against this peptide, whether of poly or monoclonal origin makes it possible to neutralize the pathogenic impact of leptospires in the evolving individual and therefore to improve the prognosis of this disease.
- the administration of a polypeptide, peptide or protein of the invention optionally in inactivated form, or of a corresponding nucleic acid or vector, makes it possible to induce a protective immune response against these infectious agents or their effects.
- the effectiveness of the peptides of the invention in the early detection of pathogenic leptospires and the induction of immune protection against these same leptospires is particularly remarkable, in particular, because the PPL protein from which they are derived does not allow '' to obtain an immunization in a direct way, without passing by the expression in an adenovirus, and that it does not allow to detect in an early and specific way the presence of strains of pathogenic leptospires in a human or animal organism.
- the peptides are generally weakly immunogenic.
- the peptide consisting of this sequence ID No. 1 is recognized in ELISA by several monoclonal antibodies against the PPL protein and in particular 4D4H4E5 and 6E5A2F2 (International Application WO01 / 59123, Example 2).
- the PP peptide is very immunogenic in the mouse, rabbit (Example 3) and gerbil (Example 7) model.
- the antibody response directed against the PP peptide was analyzed on the sera of gerbils immunized with Padenovirus-PPL. A correlation can be observed between the immune response directed against the PP peptide and the protection of animals following immunization with PPL via the adenovirus, which supports the hypothesis of a protective effect associated with the sequence of this peptide (Example 6).
- the PP peptide was coupled to the KLH carrier protein and effective protection against a virulent test was observed on the gerbil immunized with PP-KLH (Example 7).
- the PP peptide has at least a protective epitope in its sequence. The peptide alone made it possible to induce significant protection against a virulent test (example 7).
- the discriminating efficacy of the PP peptide as a diagnostic antigen for leptospirosis was evaluated on several species (humans, dogs, cattle, horses). Whatever the species considered, the PP peptide appears to be an antigen capable of demonstrating an immune response specific to infection by pathogenic leptospires, and in particular in the early phase (Example 4).
- the lipopolyosidic antigens are indeed the support for effective protection against a homologous infection, the starting point for the constitution of the vaccine preparations currently used in both humans and animals, the PP peptide, used alone, is capable of inducing cross-protection between different serogroups of the species Leptospira interrogans ss (Icterohaemorrhagiae, Canicola, Autumnalis). Heterologous protection is also induced between genomic species different from the old species Leptospira interrogans si (here L. borgpetersenii and interrogans ss), while it is absent from the old saprophytic species Leptospira biflexa si.
- the present invention therefore relates to the use of the PP peptide, of its synthetic or natural fragments, of its chemical derivatives whatever they may be, of its counterparts, of its chemical analogues, used alone or associated with other proteins or fractions lipopolyosidiques derived from leptospires, or to adjuvants of immunity whatever their nature, for the preparation of a vaccine composition for veterinary or human use, in particular for the preparation of a composition intended to induce a protective immune response against different species and serovars of pathogenic leptospires.
- the subject of the invention is also a pharmaceutical composition, in particular a vaccine, comprising a polypeptide or a nucleic acid as defined above.
- Another subject of the invention is a composition comprising one or more antibodies as described above, in particular for inducing protection.
- the immunogenic compositions, vaccines or antibodies of the invention can be used in injectable or per os or transcutaneous form, for example in association with vehicles acceptable on the pharmaceutical or veterinary level, or with adjuvants.
- the quantities of immunogens administered, and the frequency or the number of administrations can be adapted by a person skilled in the art depending on the subject, the stage of development of the injection, etc. Typically one or two injections, 2 weeks apart, are carried out, with amounts suitable for the species, for example 30 ⁇ g of peptide coupled to the carrier protein according to the invention. Additional injections may be considered, or higher amounts may be administered, particularly in the case of oral administration.
- the immunogenic compositions or vaccines of the invention have in particular the following advantages:
- Efficacy Induction of cross-protection avoiding the accumulation of different antigenic preparations of partial effectiveness in order to prevent infection by the different infecting strains present in a given country for a given species.
- a peptide of the invention avoids introducing into the vaccinated individual many antigens not necessary for protection, therefore at least useless, even dangerous during repeated use. It is thus believed that recurrent uveitis in the horse (which can lead to blindness in the animal) is induced by a post-infectious immune response due to leptospira proteins having a composition close to certain structural proteins of the horse's eye. Furthermore, it seems that in humans, repeated vaccinations are less and less well tolerated over time. In addition, no long-term pharmacovigilance data are available.
- Antibody compositions can advantageously avoid urgently and passively a serious and / or lethal development of a declared leptospirosis in humans or animals.
- Compatibility with health prophylaxis measures currently, it is impossible to differentiate post-vaccine antibodies from post-infectious antibodies, the production of agglutinating antibodies being obtained in both cases.
- This vaccine used in large production species would allow to implement a medical prophylaxis allowing despite everything the detection of the infection wild and therefore the qualifications of herds free, rewarding qualifications for export.
- the invention also relates to the polypeptides, proteins and peptides as defined above, in attenuated form, that is to say retaining the immunogenic properties and essentially devoid of other biological activity.
- the invention also relates to the use of nucleic acids or vectors as described above for the in vitro or ex vivo production of the polypeptides, proteins and peptides of the invention, whatever the methods of genetic recombination employed: transgenic animals , bacteria, eukaryotic cells, plants, plasmids, viruses, extraction of cell-free culture media, etc.
- the techniques for producing recombinant proteins are well known to those skilled in the art, and can be applied to the present invention (strong, inducible promoters, termination signals, transfection techniques, etc.).
- Figure 1 IgGi antibody response of 5 mice immunized with the PP peptide coupled to the KLH carrier protein for test 1 and 2 expressed in optical density corrected as a function of the dilution.
- FIG. 2 IgG A antibody response of 5 mice immunized with the PP peptide coupled to the KLH carrier protein for test 1 and 2 expressed in optical density corrected as a function of the dilution.
- Figure 3 Rabbit IgG antibody response immunized with the PP peptide expressed in optical density corrected as a function of the dilution.
- Figure 5 IgM and IgG antibody response of 5 sera from human patients with leptospirosis confirmed or not by MAT against the PP peptide expressed in optical density (OD).
- Figure 6 Compared response PP and PPL, either IgM or IgG, for sera from 4 SPF dogs, three of which are vaccinated against leptospirosis (CN V) expressed in optical density (OD).
- Figure 8 IgG antibody response of 4 bovine sera from suspected herds with leptospirosis against the PP peptide expressed in optical density.
- Figure 9 IgG antibody response of 6 equine sera against the PP peptide expressed in optical density.
- Figure 10 Kinetics of the antibody response against the PP peptide expressed in corrected optical density of gerbils immunized via adenovirus.
- Figure 11 Kinetics of the IgG antibody response against the PP peptide expressed in optical density corrected as a function of the dilution (A) or time (B) of gerbils immunized by the PP peptide coupled to the KLH carrier protein.
- Figure 12 Survival curve of gerbils immunized by the PP peptide coupled to the KLH carrier protein and subjected to a virulent test (EV) of leptospires of Leptospira interrogans if serovar canicola.
- EV virulent test
- Figure 13 Survival curve of gerbils immunized with PP-KLH or PP and subjected to an EN of leptospires of Leptospira interrogans if serovar canicola.
- Figure 14 Schedule of the PP peptide vaccination test coupled to the carrier protein KLH in dogs with a virulent test of Leptospira interrogans leptospires if serovar canicola
- Figure 15 Evolution of the increase in the weight of control and vaccinated dogs (immunized by the PP peptide coupled to the carrier protein KLH) before virulent test
- Figure 16 Evolution of the increase in temperature of control and vaccinated dogs (immunized by the PP peptide coupled to the protein carrier KLH) subjected to a virulent test of Leptospira of Leptospira interrogans if serovar canicola
- Figure 17 Evolution of the variation in the platelets of control and vaccinated dogs (immunized by the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of leptospires of Leptospira interrogans if serovar canicola
- Figure 18 Quantitative evolution of the white blood cell variation of control and vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira interrogans leptospires if canovola serovar
- Figure 19 Quantitative evolution of the lymphocyte variation in control and vaccinated dogs (immunized by the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of leptospires of Leptospira interrogans if serovar canicola
- Figure 20 Quantitative evolution of the variation in monocytes of control and vaccinated dogs (immunized by the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira interrogans leptospires if serovar canicola
- Figure 21 Evolution of the increase in creatinine from control and vaccinated dogs (immunized by the PP peptide coupled to the protein carrier KLH) subjected to a virulent test of Leptospira interrogans if serovar canicola
- Figure 22 Evolution of the increase in urea in control and vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira interrogators if serovar canicola
- Figure 23 Evolution of the cumulative coefficient for cultures and PCR of batches of control and vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira interrogans leptospires if serovar canicola
- Table 1 MAT and EIA titer of 5 sera from human patients with or without leptospirosis.
- Table 2 Survival table of gerbils immunized by the recombinant protein PPL with Freund's adjuvant after virulent canicola test.
- Table 3 Survival table of gerbils immunized with the recombinant protein PPL adjuvanted with aluminum hydroxide and QS 21 after virulent canicola test.
- Table 4 Mortality table of gerbils immunized with the PP peptide coupled to the KLH carrier protein after virulent test of Leptospira interrogans if canicola leptospires.
- Table 5 Table of mortality of gerbils immunized with the PP peptide coupled or not with the KLH carrier protein after virulent 10 "2 canicola test.
- Table 6 Statistical analysis of vaccination trials with the PP peptide coupled or not coupled according to the Logrank test.
- Table 7 Results of the cultures carried out on the plasma, urine and kidney samples of the 6 control dogs and the 6 vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira leptospires interrogans si serovar canicola.
- Table 8 PCR results on the plasma, urine and kidney samples of the 6 control dogs and the 6 vaccinated dogs (immunized with the PP peptide coupled with the carrier protein KLH) subjected to a virulent test of Leptospira interrogans if serovar
- the PP peptide represented in the sequence SEQ ID No. 1, is a 25 amino acid peptide which corresponds to the aal53-aal77 fragment of the PPL protein of Leptospira interrogans si
- Peptide ID No. 1 of 25 amino acids was synthesized by Neosystem.
- the crude peptide is purified by HPLC.
- the homogeneity of the purified peptide is verified by HPLC and the theoretical structure is confirmed by measurement of the mass of this peptide by ES-MS type mass spectrometry, relative to that calculated from the theoretical amino acid sequence of this peptide.
- Peptide ID No. 1 was used in ELISA. It was used as adsorption antigen (diagnostic protocol) with, as primary antibodies, different monoclonal antibodies directed against the PPL protein, in particular two monoclonal antibodies 6E5A2F2 and 4D4H4E5 and gerbil sera tested by a challenge Leptospira interrogans ss serovar canicola. The two monoclonal antibodies have recognized this peptide, which demonstrates that it has one or more immunogenic epitopes of the PPL protein. Furthermore, polyclonal gerbil sera have made it possible to demonstrate the presence of antibodies directed against this peptide in an early phase after infection with pathogenic leptospires in the gerbil. EXAMPLE 3 Immunogenicity of the PP Peptide
- the peptide used corresponds to the peptide PP (SEQ ID NO: 1) to which a cysteine is added in the N-terminal position.
- the heterobifunctional reagent, sulfo-m-Maleimidobenzoyl-N-hydrosuccimide ester (sulfo-MBS, Pierce) used allows activation of the free amino functions of the carrier protein KLH (keyhole-limpet-haemocyanin, Pierce), then coupling of the peptide via the reaction of the sulfhydryl group of cysteine added to the N-terminal end of the peptide with the maleimide part of the sulfo-MBS reagent.
- This is added in an amount of 1 mg of reagent for 10 mg of KLH, then the whole is placed under stirring for 1 h in the dark at a temperature of 15 to 25 ° C.
- the KLH protein is desalted on an NAP5 column balanced by the coupling buffer. The elution of the protein is carried out using this buffer.
- the fractions containing the activated KLH protein are combined.
- 8.5 mg of peptide in the previous buffer at a concentration of 2 mg / ml
- 10 mg of KLH concentration of the order of 5 mg / ml
- the whole is placed under stirring for 2 hours at a temperature of 15 to 25 ° C and in the dark.
- the preparation is then distributed, at a rate of 100 ⁇ g per eppendorf tube and frozen at - 80 ° C.
- mice A batch of 5 mice was formed. Two injections were administered two weeks apart subcutaneously.
- the 1st injection consists of 10 ⁇ g of PP peptide coupled to KLH with the complete Freund's adjuvant in a volume of 200 ⁇ l (v / v).
- the 2 nd injection consists of 10 mcg PP peptide coupled to KLH with incomplete Freund's adjuvant in a volume of 200 .mu.l (v / v). This test was carried out twice.
- mice receiving nothing is used as a control.
- a blood test was taken two weeks after the second injection for serological control.
- mice The anti-PP antibodies produced in mice are assayed by an ELISA technique either on individual serological samples or on pools of several sera.
- the reaction is stopped by adding an H 2 0 / DMF solution (v / v) containing 5% SDS at a rate of 100 ⁇ l per well.
- the colored reaction is read with a spectrophotometer at 405 nm.
- the results of the IgGi anti-PP antibody response expressed in OD corrected for immunization tests 1 and 2 by the PP peptide coupled to KLH are presented in FIG. 1.
- the corrected OD corresponds to the OD of the immunized animal minus the DO from pools of control animals.
- this response is very high since the sera diluted 1/8000 and 1/16000 give an OD corrected between 0.8 and 1.2 (optimum reading).
- mice except mouse 5 in test 1 There is a certain homogeneity of the IgG 2A anti-PP antibody response in mice except mouse 5 in test 1. Furthermore, this response is high but less important than the IgG1 anti-PP response. Similar results were obtained for test 2, the homogeneity of the antibody response being however lower.
- mice with the PP peptide coupled to KLH induces an anti-PP IgGi and IgG 2 A response that is both high and homogeneous.
- the PP peptide coupled to the KLH protein is therefore very immunogenic. 3-2 Immunogenicity of the uncoupled PP peptide
- the two injections were administered two weeks apart subcutaneously. Rabbit serum was collected two weeks after the second injection. Anti-PP antibodies were titrated on this serum.
- Anti-PP antibodies were assayed in rabbits by the ELISA technique described above with a biotinylated anti-rabbit antiglobulin (BioAtlantic) at 1/3000 and as a revealer of streptavidin coupled with peroxidase (RPN1231, Amersham pharmacia biotech) .
- the results of the anti-PP IgG antibody response expressed in corrected DO (DO at D28 therefore after the second immunization minus the DO at D0) of the immunization test with the PP peptide are presented in FIG. 3.
- the antibody response against the PP peptide is very high since at a dilution of 102400, a response against the PP peptide is always detectable.
- the PP peptide alone is very immunogenic.
- the PP peptide whether or not coupled to a carrier protein, is capable of inducing a strong serological response in different animal models. So the PP peptide is very immunogenic. EXAMPLE 4 Diagnostic Use of the PP Peptide
- the animal species tested are 4 in number and represent the companion or production animals most affected by leptospirosis: "the dog” the bovine
- the horse • the pig ELISA technique The anti-PP antibodies were measured by the ELISA technique described above with the antiglobulins coupled to the peroxidase chosen according to the species from which the serum analyzed came from (anti-human, anti -dog, anti-cattle, anti-horse or anti-pig (Jackson).
- the anti-PPL antibodies were assayed by the same technique. Results
- the negative control corresponds to a pool of sera from healthy individuals (before vaccination) and individually checked in MAT.
- Table 1 MAT and EIA titer of 5 sera from human patients suspected of clinical leptospirosis Analysis of the specificity of the IgM and IgG antibody responses against the PP peptide and against the recombinant PPL protein in human sera without leptospirosis
- the serum 153 272 of the confirmed lyme positive case exhibits a significant IgM serological response against the recombinant protein PPL.
- IGM directed against the PP peptide was analyzed on thirty human sera suffering from leptospirosis (from the Pasteur Institute).
- IgG and IgM dogs Vaccinated SPF dogs: Analysis of the specificity of IgM and IgG antibody responses against the PP peptide and against the recombinant PPL protein in sera from SPF dogs vaccinated or not against leptospirosis Firstly, serologies were carried out on sera from SPF dogs vaccinated against leptospirosis or not. The MAT test was carried out in parallel in the laboratory. The results of the IgG and IgM response directed against the PP peptide and against the recombinant PPL protein of these sera are shown in FIG. 6 in comparison with the negative control (pool of sera from unvaccinated SPF dogs).
- the high IgG serological response against the recombinant protein of the negative control does not allow the specific vaccine response to be estimated.
- FIG. 7A The results of the study of the IgG and IgM antibody response directed against the PP peptide of unvaccinated dogs are presented in FIG. 7A.
- the negative control corresponds to the serum of SPF dogs and does not show a significant IgM and IgG antibody response.
- Sera 210749 and 210750 were taken from the same dog 4 days apart.
- the MAT titer of the first sample is negative while the IgM anti-PP antibody response is very high (OD> 0.6) and the IgG anti-PP response begins since an OD of 0.3 is reached at the end of the evolution. clinical.
- There is a seroconversion with MAT (titer 160) and at the same time, the anti-PP IgM response decreased to give way to a very high anti-PP IgG response (OD> 0.9).
- the PP peptide can therefore prove to be very useful for the diagnosis of leptospirosis and more particularly in the early phase since an IgM anti-PP antibody response can be detected while the MAT titer is still negative.
- Serum 210845 corresponds to a dog suffering from confirmed leptospirosis. A high anti-PP IgM and IgG antibody response can be observed and of the same intensity.
- Serum 211466 corresponds to a dog with clinical signs of leptospirosis, however the MAT titer of this serum is not significant.
- the anti-PP IgM antibody response is very high (OD> 0.7) while the anti-PP IgG response is not significant. The dog is therefore most likely suffering from leptospirosis in the very early phase.
- the significant anti-PP IgM response is in favor of an initial serological response (attested by the moderate titer in MAT).
- Sera 21463 and 21457 correspond to dogs with clinical signs of Leptospirosis confirmed in MAT with a longer duration of evolution than the previous case. They exhibit a strong IgM and IgG response directed against the PP peptide.
- the PP peptide appears to be a discriminating antigen of leptospirosis infection whether dogs are vaccinated or not.
- the use of the PP antigen makes it possible to detect cases of leptospirosis in the early phase, the previous vaccine state does not prevent diagnostic interpretation unlike MAT for which the interpretation of low titers is questionable.
- Sera 421 and 1992 correspond to a serological kinetics of the same animal. During the first sample the animal was negative in MAT and became positive during the second sample, attesting that this uveitis could be interpreted as a sign of active infection in progress by leptospires.
- Animals 2077 and 6771 are slightly positive in MAT but also with respect to PP, confirm the leptospirosic origin of the uveitis observed.
- the PP peptide appears to be an antigen capable of demonstrating an immune response to an infection by leptospires, and in particular in the early phase because a response IgM anti-PP antibodies can be detected while the MAT titer is still negative or is questionable in the case of vaccinated animals.
- EXAMPLE 5 Antibody Response Against the PP Peptide of PPL Immunized Gerbils Via the Adenovirus
- a vaccination / test protocol made it possible to demonstrate the protection induced by a suspension of the recombinant Ad-ppl virus which expresses the recombinant protein PPL (International Application WO01 / 59123, Branger C, Sonrier C, Chatrenet B, Klonjkowski B, Ruvoen -Clouet N, Aubert A, Andre-Fontaine G, Eloit M. "Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination". Infect Immun. 2001 Nov; 69 (11) : 6831-8).. Anti-PP antibodies were assayed in the gerbil by the technique
- the sera were grouped on the different sampling dates D0 (before the first immunization), D21 (before the second immunization), D35 (before the virulent test) and D70 (35 days after the virulent test ).
- the anti-PP antibody response following the first injection, appears rapidly for the group immunized by the adenovirus expressing the recombinant protein PPL.
- the antibody response remained stable between 21 and 35 days ⁇ eme ⁇ eme day: the second immunization did not induce booster effect, the maximum of the vaccine response appears to be achieved as early as 21 lth day.
- test canicola effected on 35 th day allows the appearance of a response against the peptide PP in the control group and causes an anamnestic response in the group immunized by the PPL protein via adenovirus.
- An antibody response directed against the PP peptide is therefore observed following immunization with PPL via the adenovirus and this antibody response may have a role in the protection observed in this vaccinated group subjected to a canicola EN.
- Vaccination induced a maximum anti-PP response for the stimulation induced by the dose of PPL expressing adenovirus used.
- the anamnestic recovery caused by the virulent test follows a slower slope than the response developed by the witnesses.
- the PPL protein carrying the PP epitopes being secreted by pathogenic leptospires, the antigenic stimulation induced by the test virulent is weaker in animals protected by vaccination than in controls, in which the virulent strain does not encounter any obstacle to its multiplication.
- Example 6 Vaccination test with the recombinant protein PPL (comparative):
- Immunization with the recombinant protein PPL was carried out on the gerbil model in order to test the protection likely to be provided by this protein.
- Adjuvants make it possible to increase the immunogenicity of the antigens by generally creating an inflammatory reaction which slows down the elimination of the antigen and promotes its capture and its presentation to the lymphocytes. The nature of the adjuvant will guide the production of cytokines favoring the development of a cell or humoral mediated response.
- Table 2 Survival table of gerbils immunized by the recombinant protein PPL with Freund's adjuvant after virulent canicola test GRAS characters: start of gerbil mortality for each batch
- the mortality of the batches subjected to the same virulent test begins at the same time: the nth and the 12th day for the virulent tests at 100 UT respectively diluted to 10 "1 and 10 " 2 .
- Test 2 Alumina hydroxide and QS 21 adjuvant
- the immunization assay with the recombinant protein PPL was repeated under the same conditions, however the protein was adjuvanted with QS 21 and aluminum hydroxide.
- Table 3 Survival table of gerbils immunized with the recombinant protein PPL adjuvanted by aluminum hydroxide and QS 21 after virulent canicola test Characters in GRAS: start of mortality of gerbils for each batch * workforce considered from the virulent OJ test The survival rate for the batch immunized with the adjuvanted PPL recombinant protein and the control batch are similar. The mortality of the gerbils of the two lots begins on the same day: the 8 th day. It was not possible for us to demonstrate any significant protection provided by the recombinant protein PPL, whether in the presence of Freund's adjuvant or the aluminum hydroxide coupled with QS 21.
- the purpose of immunization with the PP peptide on the gerbil model is both to assess the antigenicity of the sequence chosen by the antibody response and the possible protection provided by this peptide.
- Test 1 Protocol for vaccinating gerbils with the PP peptide coupled to the KLH carrier protein
- a vaccination / test protocol has been implemented to evaluate the protection that the PP peptide (seq ID No. 1) coupled to the KLH protein can provide.
- This construct is administered subcutaneously without adjuvant with PBS as negative control (lot 2).
- Lot 2 10 gerbils, 2 injections of PBS under 300 ⁇ l The two injections in a volume of 300 ⁇ l were administered two weeks apart by the subcutaneous route.
- a virulent test by intraperitoneal injection of 0.5 ml of a canicola culture grading 90 to 100 UT (turbidimetry) diluted to 10 "2 ' 5 was carried out two weeks after the second injection.
- Antigenic response ELISA monitoring of the immune response of gerbils against the PP peptide
- Anti-PP antibodies were assayed in the gerbil by an ELISA technique (described above) with a biotinylated anti-gerbil antiglobulin (BioAtlantic) at 1/3000 and as a developer of streptavidin coupled to peroxidase (RPN1231, Amersham pharmacia biotech).
- the antigenicity of the chosen peptide is important.
- Table 4 Table of mortality of gerbils immunized by the PP peptide coupled to the protein carrier KLH after virulent test canicolalO " "' 5 .
- the mortality of lot 2 corresponding to the control group having received PBS (0/10) is very high since on day 8 no gerbil survived a virulent test of E. interrogans serovar canicola.
- This virulent challenge is severe because on one hand the DL100 was reached in 8 days and secondly mortality began early, as early as 5 ⁇ eme day, and was very quick since all the witnesses are dead in 3 days.
- Test 2 Protocol for vaccinating gerbils with the PP peptide coupled or not coupled to the KLH carrier protein
- a vaccination / trial has been implemented to assess the protection that the PP peptide (seq ID No. 1) can provide, whether or not coupled to the KLH protein.
- This construct is administered subcutaneously without an adjuvant, PBS as a negative control is administered by the same route (lot 3).
- Lot 1 12 gerbils, 2 injections of 30 ⁇ g of PP-KLH under 300 ⁇ l
- Lot 2 12 gerbils, 2 injections of 30 ⁇ g of PP in 300 ⁇ l
- Lot 3 15 gerbils, 2 injections of PBS in 300 ⁇ l
- Table 5 Table of mortality of gerbils immunized by the PP peptide coupled or not with the KLH carrier protein after virulent canicolalO "2 test .
- the mortality of batch 3, control group having received PBS, is the highest of all the batches and corresponds to the lethal dose 50 (7/15).
- the mortality in the controls started early on D6 whereas it did not start until D8 for the batch immunized with PP and D9 for the batch immunized with PP-KLH.
- the PP peptide coupled to the KLH carrier protein provides significant protection against a canicola challenge for the two experiments. This protection is significant on the survival of the animals estimated by the logrank test whatever the experience; the combination of the two experiences increases the significance of the experience, whether with the ⁇ test or the Logrank test.
- Vaccination with the PP peptide coupled or not with the KLH protein made it possible to show the protective effect of the PP peptide, twice, following a virulent test (canicola).
- Example 8 Vaccination test with the peptide PP coupled in NH 2 to the carrier protein KLH in dogs.
- the purpose of immunizing dogs with the PP peptide is to assess the protective effect provided by this peptide. Protection will be studied under two components: clinical protection but also protection against renal carriage.
- This construct is administered subcutaneously without adjuvant with PBS as negative control (lot 2).
- the Virulent Test was performed on D 0 .
- the animals receive a virulent test at the rate of a virulent culture of 4 to 6 days.
- each animal receives 0.3 ml in each eye of the culture titrating 120 UT (that is 2 ⁇ 10 8. Leptospires per ml) and intraperitoneally 4 ml of the same culture diluted to the tenth.
- BALANCE SHEET
- the test phase took place from Ji to D 41 , with the test on D 0 . During this phase, the temperature was measured at Jo, Ji, h, h, h,,, -
- the haematological parameters were measured on D- 1; J 3 , J, J ÎO , J 1 , Jis and
- the evolution of the lymphocytes between D ⁇ and D 41 is shown in FIG. 19. Significant lymphopenia is noted on D 4 in the controls, perfectly unnoticed in the vaccinated in which only post-infectious lymphoproliferation is observed.
- Period of acute renal involvement (Jo-Jio): no significant difference between controls and vaccinated despite a tendency (p ⁇ 0.15) to a more intense expression in the controls (higher urea)
- the presence of leptospires was studied jointly by PCR (PCR specific for pathogenic leptospires) and by culture in the urine on D 0 , D 10s D 1 , J ⁇ 8 , D25, D41 and in the kidney samples of each animal at the end of the test on D 41 .
- the PCR results are shown in Table 8.
- the bacteriological results were quantified thanks to the implementation of a coefficient: by immediate culture.
- a single living leptospire can give a positive culture.
- a relative estimate of the bacterial concentration is expressed by a coefficient of 3 when a culture has developed in the third serial dilution made from the sample. by PCR on frozen samples.
- a positive PCR does not prejudge the viability of the bacteria detected and, moreover, a PCR is only positive for 10 leptospires present.
- a coefficient of 1 was assigned to a PCR with a weak signal and a coefficient of 2 to a strongly positive PCR.
- the urea and creatinine levels are lower in the vaccinated during the septicemia phase than those observed in the controls.
- Renal colonization detected later in vaccinated dogs than in control dogs corresponds to colonization with lower noise or later.
- the fact of simultaneously observing a significant increase in urea in control dogs is in favor of colonization at a lower level in the vaccinated.
- Table 7 Results of the cultures carried out on the plasma, urine and kidney samples of the 6 control dogs and the 6 vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira leptospires interrogans if serovar canicola
- Table 8 PCR results on plasma, urine and kidney samples from 6 control dogs and 6 vaccinated dogs (immunized with the PP peptide coupled to the carrier protein KLH) subjected to a virulent test of Leptospira interrogans leptospires if serovar canicola
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Abstract
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Application Number | Priority Date | Filing Date | Title |
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FR0213585 | 2002-10-30 | ||
FR0213585A FR2846658B1 (fr) | 2002-10-30 | 2002-10-30 | Peptides pour la prevention, le diagnostic et le traitement de la leptospirose animale et/ou humaine. |
PCT/FR2003/003154 WO2004041855A1 (fr) | 2002-10-30 | 2003-10-24 | Peptides pour la prevention, le diagnostic et le traitement de la leptospirose animale et/ou humaine |
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EP1576003A1 true EP1576003A1 (fr) | 2005-09-21 |
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EP03780234A Withdrawn EP1576003A1 (fr) | 2002-10-30 | 2003-10-24 | Peptides pour la prevention, le diagnostic et le traitement de la leptospirose animale et/ou humaine |
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US (1) | US7635480B2 (fr) |
EP (1) | EP1576003A1 (fr) |
AU (1) | AU2003288330B2 (fr) |
FR (1) | FR2846658B1 (fr) |
WO (1) | WO2004041855A1 (fr) |
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WO2014093619A1 (fr) | 2012-12-12 | 2014-06-19 | Immport Therapeutics, Inc. | Procédés et compositions d'antigènes de protéine pour le diagnostic et le traitement de la leptospirose |
US9850278B2 (en) | 2013-04-25 | 2017-12-26 | Carmel-Haifa University Economic Corp. | Synthetic anti-inflammatory peptides and use thereof |
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US6077822A (en) * | 1993-09-14 | 2000-06-20 | Dumex-Alpharma A/S | Drug salts |
US5837263A (en) * | 1995-05-19 | 1998-11-17 | The Regents Of The University Of California | Leptospira membrane proteins |
US6306623B1 (en) * | 1998-02-24 | 2001-10-23 | The University Of California | Leptospiral major outer membrane protein LipL32 |
FR2804685B1 (fr) * | 2000-02-08 | 2004-08-13 | Envt Toulouse | Utilisation d'une proteine de leptospire pour la prevention et/ou le diagnostic et/ou le traitement de la leptospirose animale et/ou humaine |
GB0003217D0 (en) * | 2000-02-11 | 2000-04-05 | Yaba Ltd | Invention |
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2002
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- 2003-10-24 AU AU2003288330A patent/AU2003288330B2/en not_active Ceased
- 2003-10-24 US US10/533,193 patent/US7635480B2/en not_active Expired - Fee Related
- 2003-10-24 WO PCT/FR2003/003154 patent/WO2004041855A1/fr not_active Application Discontinuation
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FR2846658B1 (fr) | 2007-03-30 |
AU2003288330B2 (en) | 2010-05-13 |
WO2004041855A1 (fr) | 2004-05-21 |
AU2003288330A1 (en) | 2004-06-07 |
FR2846658A1 (fr) | 2004-05-07 |
US20070197432A1 (en) | 2007-08-23 |
US7635480B2 (en) | 2009-12-22 |
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