Immunoglobulin against the anthrax toxin

Abstract

The present invention relates to a class-G immunoglobulin against the anthrax toxin protective antigen (PA), or one of the fragments of same, comprising at least: a variable heavy-chain region comprising an amino acid sequence represented by the sequence SEQ ID NO: 1, or comprising an amino acid sequence having at least 90% identity with the sequence SEQ ID NO: 1, and comprising the amino acids Leucine in position 51 and Glycine in position 67, and a variable light-chain region comprising an amino acid sequence represented by the sequence SEQ ID NO: 2, or comprising an amino acid sequence having at least 90% identity with the sequence SEQ ID NO: 2, and comprising a Leucine amino acid in position 55. The invention also relates to the uses of such an immunoglobulin.

Claims

1. A class-G immunoglobulin directed against the anthrax toxin protective antigen, wherein each heavy chain of the immunoglobulin comprises, or consists of, SEQ ID NO:5, and each light chain of the immunoglobulin comprises, or consists of, SEQ ID NO:6.

2. The immunoglobulin of claim 1, wherein said heavy chain and/or said light chain is bonded to a signal peptide.

3. The immunoglobulin of claim 1, wherein: each of the heavy chains comprises SEQ ID NO:7, and each of the light chains comprises SEQ ID NO:8.

4. The immunoglobulin of claim 1 having on its Asn297 glycosylation site N-glycans having a degree of fucosylation less than 65%.

5. The immunoglobulin of claim 4, having on its Asn297 glycosylation site a glycan structure of biantennary type, with short chains and a low degree of sialylation, having non-intercalated end mannoses and/or end N-acetylglucosamines.

6. The immunoglobulin of claim 5, having a content of greater than 60% for G0+G1+G0F+G1F forms, wherein G0F+G1F forms are less than 50%.

7. The immunoglobulin of claim 5, having a content of greater than 60% for G0+G1+G0F+G1F forms, and wherein fucose content is less than 65%.

8. The immunoglobulin of claim 5, having a content of less than 40% for G1F+G0F forms.

Description

FIGURE LEGENDS

(1) FIG. 1

(2) Pearl necklace diagram of the variable region of the heavy chain (A) and of the light chain (B) of the 35PA83 immunoglobulin.

(3) The pearl necklace IMGT representation is produced in accordance with IMGT numbering. The hatched circles correspond to the missing positions of the IMGT numbering.

(4) FIG. 2

(5) Pearl necklace diagram of the variable region of the heavy chain (A) and of the light chain (B) of the 35PA83 6.20 immunoglobulin.

(6) The pearl necklace IMGT representation is produced in accordance with IMGT numbering. The hatched circles correspond to the missing positions of the IMGT numbering.

(7) FIG. 3

(8) HILIC-UPLC/FD profile of the N-glycans of the 35PA83 6.20 antibody, released after treatment using peptidyl-N-glycosidase F (PNGase F).

(9) The X-axis corresponds to the elution time in minutes. The Y-axis indicates the intensity noted for each compound identified, in emission units.

EXAMPLES

Example 1: Construction of the Vector Encoding the 35PA83 6.20 Immunoglobulin

(10) Escherichia coli Strains

(11) The following E. coli strains were used: XL1 (Stratagene, La jolla, CA): recA1, endA1, gyrA96 thi-1 hsdR17 sup E44 relA1 lac [FproAB laclqZM15 Tn10(Tetr)]. SURE (Stratagene): e14(McrA) (mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [FproAB laclqZM15 Tn10 (Tetr)]. HB2151 (Carte et al., 1985), used for the expression of soluble Fabs. TOP 10 (Invitrogen): used for constructing the eukaryotic expression vector.
Toxins

(12) The anthrax toxins (PA83, LF and EF) were purchased from List laboratories.

(13) Construction of Mutant Fabs from 35PA83

(14) A variant was first constructed in order to humanize the 35PA83 immunoglobulin fragment. This variant was obtained by carrying out the mutations described in the tables below:

(15) TABLE-US-00004 TABLE 4 Mutations for humanization of the 35PA83 heavy chain Amino acid position 35PA83 Hu35PA83 1 None Q 2 None V 3 None Q 4 None L 5 None Q 6 None E 7 None S

(16) TABLE-US-00005 TABLE 5 Mutations for humanization of the 35PA83 light chain Amino acid position 35PA83 Hu35PA83 1 None A 2 None I 3 None Q 4 None L

(17) Using this humanized variant, mutant sequences derived from the 35PA83 gene were then generated by a circular mutagenizing amplification technology, Massive Mutagenesis (Biomthodes, Evry, France). The mutations were introduced into the CDRs of the heavy and light chains using NNS codons (N encodes A, T, G or C, and S encodes G or C). The CDR regions were defined according to Kabat et al. (Wu and Kabat, 1970) and IMGT (Lefranc, Pommie et al., 2003).

(18) The DNA obtained was used to transform SURE cells by electroporation. After addition of SB medium supplemented with carbenicillin to the culture and incubation for 1 h at 37 C., 1 ml of VCSM13 helper phage (Andris-Widhopf et al., 2001) (about 1012 pfu) was added to the culture. After incubation for 2 h, 70 g/ml of kanamycin were added and the culture was shaken over night.

(19) Selection of Immunoglobulins by Phage Display

(20) The phage-immunoglobulin particles were purified and concentrated from 50 ml of culture by precipitation with polyethylene glycol (PEG), then resuspended in 3 ml of PBS-1% BSA-0.02% azide and filtered on a 0.45 m filter. The titer of this phage preparation was approximately 1010 pfu/ml. The phage-immunoglobulins were subjected to three infection-selection-recovery cycles, corresponding to 5, 10 and 15 washes respectively, as previously described (Andris-Widhopf, Rader et al., 2000).

(21) Expression, Periplasmic Extraction and Purification of Soluble Mutant Fabs

(22) Each DNA variant was transformed in bacteria of the E. coli strain HB2151, rendered chemically competent. The cells were cultured at 30 C., and shaken at 250 rpm in 1 l of SB medium containing 50 g/ml of carbenicillin and 0.1% of glucose. When the culture reached an absorbance at =600 nm of 1.5, induction with 1 mM of IPTG (isopropyl -D-1-thiogalactopyranoside) was carried out for 18 h at 22 C.

(23) The immunoglobulin fragments were extracted with polymyxin B sulfate (Sigma) and purified on a nickel column (Ni-NTA spin column, Qiagen, Valencia, Calif.) according to the manufacturer's instructions, then dialyzed with 1PBS at 4 C. for 3 h.

(24) Quantification of the Soluble Fab

(25) The purity of the Fab was tested by SDS-PAGE and its concentration was determined using the Quantity One software (Biorad).

(26) Real-Time Measurement of Surface Plasmon Resonance (SPR)

(27) The kinetic constants for the interaction between PA83 and the 35PA83 variants were determined using the Biacore X SPR system (BIAcore, Uppsala, Sweden). The PA83 was immobilized on a CM5 sensitive chip (Biacore) using a procedure for coupling of amines by injection of 30 l of 2 g/ml of PA83 in 10 mM of sodium acetate, pH 4.5. To minimize the probability of rebinding, the K.sub.D was measured using a high flow rate (30 l/min) and a minimum amount of coupled antigen (approximately 500 RU, resonance units). The degree of binding of various Fab concentrations ranging from 5 to 400 nM in PBS was determined at a flow rate of 30 l/min. The binding data was introduced into a 1:1 langmuir model of the BIA evaluation software (Biacore). The association and dissociation constants (k.sub.on and k.sub.off respectively) for the binding of the Fab to PA83 were determined at 35 C.

(28) Sequence Analysis

(29) The sequences of the heavy and light chains of the clones selected were determined by Genome Express (Meylan, France) using the ompseq and newpelseq primers (Andris-Widhopf, Rader et al., 2000). The sequences were analyzed on line, using the IMGT system.

(30) Among the clones selected, the 35PA83 6.20 variant was identified.

(31) The heavy-chain variable region (VH) of the 6.20 variant (SEQ ID No.: 1) has 2 mutations compared with that of the humanized 35PA83 Fab: H-L (residue 54 according to the IMGT nomenclature) and S-G (residue 74 according to the IMGT nomenclature).

(32) The light-chain variable region (VL) of the 6.20 variant (SEQ ID No.: 2) has 1 mutation compared with that of the humanized 35PA83 Fab: Q-L (residue 68 according to the IMGT nomenclature).

(33) Construction of the ATH-GA Expression Vector for Expression of the 35PA83 6.20 Immunoglobulin

(34) A sequence encoding the MB7 optimized signal peptide

(35) ##STR00001##
was added in the N-terminal position of the two sequences encoding the variable parts of, respectively, the heavy chain and the light chain of the 35PA83 6.20 variant (SEQ ID Nos: 7 and 8). These sequences were optimized and synthesized by GeneArt (Regensburg, Germany).

(36) The sequences of the heavy and light chains of the variant were then cloned into the HK gen EFss vector in order to obtain the ATH-GA vector.

(37) The sequencing of the ATH-GA vector was carried out according to the Sanger technique (or chain termination method, ref.: Sanger F. et al, 1977, PNAS 74: 5463). The sequencings were carried out by Eurofins MWG Operon (Ebersberg, Germany) according to the GLP regulatory quality level. This involves the maximum quality level, with double-stranded coverage of the DNA sequence, a 2-fold minimum redundancy, accuracy greater than 99.999%, dedicated instruments, the drafting of a quality report and archiving of the documents generated.

(38) The ATH-GA vector preparation was stored in TE buffer (10 mM Tris pH 8 and 1 mM EDTA) at 20 C. before adjustment to the concentration of 1 g/l for transfection into the YB2/0 cell lines.

Example 2: Obtaining of Transformants Producing the 35PA83 6.20 Immunoglobulin

(39) The 35PA83 6.20 antibody was produced in the YB2/0 lines. For the experiments below, the ELISA technique implemented is carried out according to the following conditions:

(40) 96-well microtitration plates (maxisorp, Nunc, Denmark) are coated with PA diluted in PBS (5 g/ml, 100 l per well), over night at 4 C. The plates are blocked by adding 200 l of PBS-5% BSA at 37 C. for 1 hour, and sera serially diluted in PBS-0.1% Tween 20-1% BSA are incubated (100 l per well) at 37 C. for 2 hours. An anti-mouse IgG alkaline phosphatase conjugate or an anti-human IgG alkaline phosphatase conjugate (Sigma) is incubated (1/10 000) at 37 C. for 1 hour. A p-nitrophenyl phosphate substrate is then incubated for 30 minutes at ambient temperature. The results are determined by measuring the absorbance at 405 nm with an automated microplate reader (iEMS reader MF, Labsystems, Helsinki, Finland). The final dilution point, the reversion of which determines the titer of the serum, is determined as giving a signal less than or equal to 2 times the naive serum used for the negative control.
a. Transformation Level The ATH-GA vector was introduced into the YB2/0 host cell line by electroporation. After selective culturing (with the G418 selective agent), pools of transfectants were obtained and plated out in a semi-solid medium in the presence of fluorescent anti-human IgG antibody and under conditions allowing the growth of isolated colonies. The fluorescence intensity of the colonies, proportional to their production capacity, was analyzed using the ClonePixFL automated device (CPFL) and the colonies exhibiting the greatest fluorescence were sub-cultured by the automated device.
b. Transformant Selection Production level: first screening of transformants which are stronger producers

(41) The production of human IgGs was determined by the ELISA technique on the supernatants of the double-selection P96 wells containing the cells in order to perform a first hierarchization of the cloids with regard to their production capacities.

(42) Three successive screenings (every 2-3 days) were carried out and the 10 best producers of each screening were selected. Out of 528 transformants, 27 were pursued and maintained in P24 and a study of their productivity at D+3 and of their maximum production (D+7) was carried out in parallel. Productivity at D+3 and maximum production (D+7)

(43) The best producer clones selected with a productivity mostly greater than 5 pcd and a maximum production greater than 10 g/ml were subjected to cell amplification in selective medium (double selection) for saving in liquid nitrogen.

(44) c. Selection of a Clone and Production of the 35PA83 6.20 Immunoglobulin in a Cell Cultivator

(45) A clone was retained for the production of the 35PA83 6.20 immunoglobulin in a cell cultivator (10 L) as a function of its growth and productivity characteristics.

(46) The 35PA83 immunoglobulin was produced, concentrated and purified.

Example 3: N-Glycosylation Profile of 35PA83 6.20

(47) An assay of the fucose level was carried out by the ELISA technique on the supernatants of the cloids selected at D+3 and D+7.

(48) In order to determine the N-glycosylation profile of the 35PA83 6.20 immunoglobulins, the latter were treated enzymatically using peptidyl-N-glycosidase (PNGase F). Quantitative profiling of the N-glycan mixture was then carried out by ultra performance liquid chromatography in HILIC mode (UPLC/FD).

(49) Results:

(50) FIG. 3 shows the N-glycosylation profile of the 35PA83 6.20 immunoglobulins. Table 6 compiles a list of the major species identified, modified by their relative molar ratio (RMR, %), the degree of fucosylation (Fuc), the level of intercalated GlcNAc (Bis-GlcNAc) and the degree of sialylation, and also the galactosylation index (Gal).

(51) TABLE-US-00006 TABLE 6 Glycan Abbreviations structures G0-Gn 0.7 G0F-Gn 0.7 G0 26.4 G0B 10.7 G0F 26.2 Man5 0.0 G0FB 15.8 G1(1,6) 2.7 G1(1,3) 1.5 G1(1,6)B 1.4 G1(1,3)B 0.0 G1(1,6)F 4.4 G1(1,3)F 1.9 G1(1,6)FB 3.3 G1(1,3)FB 0.3 G2 0.0 G2B 0.2 G2F 0.6 G2FB 1.5 G1(1,3)NeuAc1 0.0 G1(1,3)FNeuAc1 0.3 G1(1,3)FBNeuAc1 0.3 G2(1,3)NeuAc1 0.0 G2(1,3)BNeuAc1 0.0 G2F(1,3)NeuAc1 0.0 G2FB(1,3)NeuAc1 0.4 G2NeuAc2 0.0 G2BNeuAc2 0.0 G2FNeuAc2 0.0 G2FBNeuAc2 0.0 Unidentified peak 0.6 Degree of fucosylation (%) 40 Galactosylation index (%) 22 Level of Bis-GlcNAc (%) 34

(52) The 35PA83 immunoglobulin is characterized by a glycosylation profile which is strictly of biantennary complex type with predominantly agalactosylated structures of G0 type (80%) the pentasaccharide nucleus of which is possibly substituted with a fucose residue (G0F), an intercalated GlcNAc residue (G0B), or both (G0FB). Mono- and bigalactosylated structures of G1,2(F)(B) type which may or may not be sialylated are also observed in low abundance (80%), with an N-glycan galactosylation index of 22%. The degree of fucosylation of the N-glycans is 40%, while the level of intercalated GlcNAc structures is 34%.

Example 4: Measurement of the Kinetic Constants of the 35PA83 6.20 Immunoglobulin

(53) After the production of 35PA83 6.20 immunoglobulin in YB2/0 cells, the cell culture supernatant is recovered, concentrated 15 times, and then subjected to affinity chromatography by means of a recombinant protein A-Sepharose. A second purification step is carried out by means of the HiPrep 16/10 SP FF cation exchange column. The integrity of the immunoglobulin purified and the absence of contaminant are verified by SDS-PAGE and by ELISA for the binding to recombinant PA83.

(54) The affinity constants are measured by surface plasmon resonance (SPR) by means of the Biacore SPR Systems (Biacore Uppsala, Sweden). PA83 (List Biological Laboratories, Inc., Campbell, Calif.) is immobilized at a maximum of 210 RU on a CM5 chip (Biacore SPR Systems) by means of an amine bond, according to the supplier's instructions. A flow of 30 l/min is maintained during the measurement. For each measurement, a minimum of 6 dilutions of immunoglobulin in HBS-EP buffer (Biacore SPR Systems), with concentrations of between 10 and 0.1 g/ml, are tested for 1900 seconds. After each immunoglobulin dilution, the chip is regenerated with glycine, pH 1.5 (Biacore SPR Systems), with a flow of 10 l/min for 30 seconds. The affinity constants are calculated by means of a bivalent analyte method (Karlsson et al. 1991), and verified with tests of internal consistency (Schuck et al. 1996).

(55) Results:

(56) The 35PA83 6.20 immunoglobulin exhibits, with respect to the PA83 antigen, an association constant (Ka) of 2.9310.sup.5 M, a dissociation constant (K.sub.d) of 9.7010.sup.6 M and an affinity constant (K.sub.D) of 3.310.sup.11 M.

Example 5: Measurement of the Neutralizing Effect of 35PA83 6.20

(57) The in vitro neutralization test is carried out according to the protocol described by Little et al. (Little et al., 1990). The J774A.1 mouse macrophage cell line (ATCC-TIB67) is cultured in untreated culture flasks in order to grow in suspension. The cells are sub-cultured twice a week in IMDM medium, 10% FCS, at 0.310.sup.6 cells/ml.

(58) The toxic activity is obtained by extemporaneously mixing PA (List Biological Laboratories, Inc.) and LF (List Biological Laboratories, Inc.). A cell suspension at 510.sup.5 cells/ml in IMDM without phenol red, containing 5% of FCS, is distributed into a round-bottomed microplate in a proportion of 200 l/well. After 16 h at 37 C., 5% CO.sub.2, the adherent cells form a carpet of cells in monolayer.

(59) D0:

(60) Elimination of the culture medium (elimination of the LDH spontaneously released during the culture). Preparation of the concentration ranges of PA 100 ng/ml constant+LF variable concentration (100-50-25-12.5-6.25-3.12-1.6-0.78 ng/ml). Preparation of the concentration ranges of LF 100 ng/ml constant+PA variable concentration (100-50-25-12.5-6.25-3.12-1.6-0.78 ng/ml). 200 l of each of the toxin mixture concentrations are distributed per well, the plate is incubated for 4 h at 37 C. In order to test the neutralizing capacity of the anti-PA antibody, a fixed concentration of toxic mixture is chosen and is pre-incubated for 1 h at 37 C. with a series of dilution of the antibody, then distributed into the plate. A range of cell lysis induced by 1% Triton X100 and representing 100-75-50-25-12.5-6.25 and 0% lysis.

(61) The samples are deposited in triplicate.

(62) D0+4h:

(63) The concentration of LDH released by the lysis of the cells is measured according to the instructions of the Cyto Tox 96 Non-radioactive Cytotoxic Assay kit from Promega.

(64) The optimal concentrations of PA and LF are determined by carrying out a dose-effect of PA in the presence of a fixed concentration of LF and a dose-effect of LF in the presence of a fixed concentration of PA.

(65) The EC50 values are, for PA, 31 and 32 ng/ml in the presence of 100 ng/ml of LF, and, for LF, 6.5 and 6.2 ng/ml in the presence of 100 ng/ml of PA.

(66) The combination of 100 ng/ml of PA with 100 ng/ml of LF makes it possible to obtain a maximum cell lysis close to 80%.

(67) The neutralizing activity of the anti-PA antibody is determined by its capacity to bind to PA and to thus block the binding of PA to the cell receptors.

(68) The toxin, consisting of the mixture of 100 ng/ml of PA and 100 ng/ml of LF, is incubated in the presence of various concentrations of anti-PA antibody for 1 h at 37 C. 200 l of the mixture are then deposited in triplicate in the wells containing the J774A.1 cells.

(69) Results:

(70) The neutralizing effect of the 35PA83 6.20 antibody was evaluated in 2 series of tests under the following conditions:

(71) Series 1: anti-PA antibody concentration: 50-10-2-0.4-0.08-0.016-0.0032-0 ng/ml.

(72) Series 2: anti-PA antibody concentration: 50-15.01-4.51-1.35-0.406-0.1220-0.037-0 ng/ml.

(73) Under these conditions, the anti-PA neutralizes the effect of the PA/LF toxin in a dose-dependent manner and completely inhibits cell mortality at concentrations above 10 ng/ml.

(74) The 50% neutralization value (EC.sub.50) is determined for the 35PA83 6.20 immunoglobulin; it is around 4-5 ng/ml.

Example 6: Pharmacokinetic Studies

(75) In order to evaluate the half-life time of the 35PA83 6.20 immunoglobulin, six six-week-old A/J mice (Harlan, Gannat, France) were divided up into two subgroups of equal size. All the mice received the 35PA83 6.20 immunoglobulin, administered by means of a single subcutaneous injection at the dose of 10 mg/kg. The blood was collected by daily retro-orbital puncture, from day 1 and up to day 6 after injection, and then from day 8 up to day 10 after injection, alternating between the mice on each separate day. The half-life time of the 35PA83 6.20 immunoglobulin was established on the basis of the results of the ELISA assays carried out on the pools of serum samples, after linear extrapolation of the values obtained.

(76) In order to carry out the ELISA assays, the wells of 96-well microtitration plates were coated by incubation with the PA83 antigen or the LF antigen (List Biological Laboratories, Inc.) diluted in PBS buffer (5 g/ml, 100 l per well) over night at 4 C. The free sites of the microplate wells were then blocked by incubation with a volume of 200 l of a solution of bovine serum albumin (BSA) at 5% in PBS buffer, for 1 hour at 37 C. The sera were serially diluted in a PBS buffer containing 0.1% of Polysorbate 20 (Tween 20) and 1% of BSA, then incubated in the plates (100 l/well) for 2 hours at 37 C. The wells of the plates were then incubated with an anti-mouse IgG/alkaline phosphatase conjugate or an anti-human IgG/alkaline phosphatase conjugate diluted to 1/10 000 (Sigma, Saint Louis, Mo., United States), for 1 hour at 37 C. The p-nitrophenyl phosphate substrate (Sigma) was then added and the plates were then incubated for 30 minutes at ambient temperature. The absorbance at 405 nm was determined using an automatic microplate reader (iEMS Reader MF, Labsystems, Helsinki, Finland). The limiting dilution point, the reciprocal value of which corresponds to the antibody titer of the serum, was defined as the point for which the value of the signal was equal to double the value of the signal measured for the serum of naive mice. The serum of naive mice is used as a negative control.

Example 7: Study of Passive Protection of Rats

(77) For the in vivo trials, Fischer rats (250 to 300 g) (C. River, L'Abresle, France) are injected with 40 g of PA (List Biological Laboratories, Inc., Campbell, Calif.) and 8 g of LF per 250 g of rat, in the manner described in Ezzell et al. (Ezzell et al., 1984), except for the fact that the tail vein is used. 4 animals are used per group and, for the evaluation of the 35PA83 6.20 immunoglobulin, the immunoglobulin is added to the PA and to the LF before the injection. The rats are observed twice a day for 10 days. All the in vivo trials presented in this study are approved by the local ethics committee for animal experiments and animal care.

(78) Preparation and Use of Sterne Spores:

(79) Spores of B. anthracis Sterne (Pasteur collection) are prepared as set out in Albrecht et al. (Albrecht et al., 2007) and kept frozen (20 C.). The spores are counted by viable plate counting after freezing/thawing and the count is verified when each tube is used in this study. The LD50 of these spores administered intravenously to 9-week-old male A/J mice (Harlan, Gannat, France), weighing 20-25 g, is established at 110.sup.4, leading to death in 48 to 72 hours, that is to say close to a value of 210(4) used in another study (Albrecht et al., 2007).

Example 8: Prophylaxis Using the 35PA83 6.20 Immunoglobulin, Short Treatment with Tetracycline, or Both

(80) For the studies of a prophylactic scheme of the 35PA83 6.20 immunoglobulin or using tetracycline only, the immunoglobulins are injected into groups of 10 A/J mice, subcutaneously, 12 hours before the infection (an injection of 5 mg/kg or of 2 mg/kg). The challenge is administered as 10 000 LD.sub.50 or 110.sup.8 spores and the mice are observed twice a day for 2 weeks, then 5 times a week for an additional 2 weeks. The surviving mice are retested by infection one month later, with the same amount of spores, and observed for an additional month. For the studies of a prophylactic scheme involving both tetracycline and the 35PA83 6.20 immunoglobulin, groups of 10 mice are treated with tetracycline as in the scheme involving tetracycline alone, but, in addition, the 35PA83 6.20 immunoglobulin is injected 12 hours before the challenge. For the active protection studies, 10 mice are injected subcutaneously with 5 g of PA per mouse, in complete Freund's adjuvant. A second group receives the same injection and then, 1 month later, the immune response of this group is stimulated with the same dose of PA in incomplete Freund's adjuvant.

Example 9: Prophylaxis Using the 35PA83 6.20 Immunoglobulin, Short Treatment with Doxycycline, or Both

(81) The study of prophylaxis with doxycycline, with or without the 35PA83 6.20 immunoglobulin, was carried out with groups of ten 10-week-old A/J mice (Harlan, Gannat, France), which were injected prophylactically with the antibiotic intraperitoneally, at the single daily dose of 5 mg/kg. The chemoprophylaxis was begun 12 hours before the infection and was carried out for 7 days, thereby representing a 9/10 reduction in the standard duration, which is 60 days.

(82) A doxycycline dosage which is approximately double the standard human dosage was chosen (daily dosage of 3 mg/kg for an adult human), and it has been shown that smaller doses are effective against B. anthracis (Friedlander et al., 1993, J Infect Dis, Vol. 167: 1239-1243; Kalns et al., 2002, Biochem Biophys Res Commun, Vol. 297: 506-509). Larger doses have been used (Heine et al., 2007, Antimicrob Agents Chemother, Vol. 51: 1373-1379); it was, however, observed that a dose of 50 mg/kg appeared to be poorly tolerated in the A/J mice, which then exhibited swelling of the abdomen and hair standing on end. In order to supplement the doxycycline treatment with the 35PA83 6.20 immunoglobulin, a single dose of this antibody (1 or 2 mg/kg) was optionally injected concomitantly with the final dose of doxycycline. The infection used 110.sup.8 intraperitoneally injected spores, which represents 10 000 LD.sub.50. The mice were observed twice a day for the first two weeks, then five times per week for an additional two weeks.

Example 10: Therapy with the 35PA83 6.20 Immunoglobulin, Short Treatment with Ciprofloxacin, or Both

(83) For the therapeutic scheme studies, groups of 10 A/J mice are challenged with a dose of 1000 LD50 or 110.sup.7 spores. After 12 hours, the 35PA83 6.20 immunoglobulin (subcutaneously, 1 injection of 10 mg/kg) or the ciprofloxacin (subcutaneously, 50 mg/kg twice a day for 5 days) is injected separately or the ciprofloxacin and the 35PA83 6.20 immunoglobulin are both injected on the first day, and then the ciprofloxacin alone is again injected for 4 additional days.

Example 11: Therapy with the 35PA83 6.20 Immunoglobulin, Short Treatment with Ciprofloxacin, or Both (Other Trial)

(84) For the curative treatment studies, groups of 10 A/J mice are infected with a dose of 1000 LD.sub.50 or 110.sup.7 spores. After 12 hours, the mice were treated with ciprofloxacin (subcutaneously, with an initial injection of 25 mg/kg) or with the 35PA83 6.20 IgG (subcutaneously, 1 injection of 10 mg/kg) separately; or ciprofloxacin and the 35PA83 6.20 IgG are both injected simultaneously at two different sites. Additional periods of 24 hours and 48 hours before beginning the combined treatment (ciprofloxacin and 35PA83 6.20 IgG) were also tested. After the first administration of the treatment, ciprofloxacin alone (25 mg/kg, twice a day) was injected for the following 4.5 days. The ciprofloxacin dose was chosen to be approximately double the standard dose in humans (daily dose of 20 mg/kg in adult humans), this dose having already been used effectively against B. anthracis (Kalns et al., 2002, Biochem Biophys Res Commun, Vol. 297: 506-509). The tolerance at this selected dose was favorably tested in the A/J mice before beginning this study. This part of the study aims essentially to solve the problem of short-term survival following a delayed treatment, and the monitoring was limited to the period of 18 days following the infection.

Example 12: Comparison Between the Passive and Active Prophylactic Anti-Anthrax Treatments

(85) A passive prophylactic anti-anthrax treatment consists of a treatment with the 35PA83 6.20 immunoglobulin. An active prophylactic anti-anthrax treatment consists of a treatment by immunization with the PA antigen.

(86) In order to compare the active and passive immunoprotection, a group of ten mice was immunized subcutaneously with 5 g of PA83 in complete Freund's adjuvant and infected intraperitoneally with 10 000 LD.sub.50, one month later. Another group of ten mice was immunized in an identical manner, but received a booster immunization four weeks later with 5 g of PA83 in incomplete Freund's adjuvant, and infected one month after the second injection. In parallel, the passive protection by the 35PA83 6.20 immunoglobulin against the same infection was evaluated. All the infected animals were observed for one month, and the results of the two types of prophylaxis were compared.

Example 13: Comparison Between a Passive Immunization and a Late Treatment with the 35PA83 6.20 IgG Only in White New Zealand (WNZ) Rabbits Infected with Spores of 9602

(87) For the passive immunization study, the 35PA83 6.20 IgG is injected intravenously at 2.5, 1 and 0.5 mg/kg in 3 groups of 8 WNZ rabbits, anesthetized beforehand with the anesthetic IMALGENE1000 (Merial, Lyon, France). Five minutes later, the animals are brought into contact with 25 l of a suspension of spores of the B. anthracis virulent strain 9602, deposited on each nostril for inhalation into the lungs, and corresponding to 100 LD.sub.50.

(88) For the late treatment, the same experimental conditions were used, except that 2 groups of 8 animals receive the injection of IgG (2.5 mg/kg) 6 h after being brought into contact with 80 LD.sub.50 or 200 LD.sub.50 of B. anthracis 9602 spores.

(89) For each group, 4 additional animals are used under the same experimental conditions, as positive controls. All the experiments with the B. anthracis 9602 strain are carried out in a security level 3 laboratory, and the animals are observed 21 days after the bringing into contact.

LITERATURE REFERENCES

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