Preparation of recombinant human plasma phospholipid transfer protein (PLTP) from the milk of transgenic rabbits

Abstract

The invention relates to obtaining a preparation of recombinant human PLTP from the milk of a transgenic animal containing in its genome one or more copies of a transgene comprising a polynucleotide coding for human PTLP, placed under transcriptional control of a promoter permitting its specific expression in the cells of the mammary glands of said animal. The recombinant human PLTP preparation obtained can be used in the prevention or treatment of septic shock.

Claims

1. A method of producing an active recombinant human plasma phospholipid transfer protein (PLTP) preparation, said method comprising (i) harvesting milk produced by a transgenic rabbit secreting active human PLTP in its milk, (ii) precipitating casein in the harvested milk in the presence of EDTA at acidic pH of less than or equal to 4.6, (iii) clarifying the product obtained in (ii) and recovering the whey, and (iv) extracting the PLTP from the whey to form the PLTP preparation; wherein the PLTP preparation has a phospholipid transfer activity at least 50 times higher than in untreated milk produced by a transgenic animal that secreted active human PLTP in its milk.

2. The method as claimed in claim 1, wherein the extraction of the PLTP from the whey is carried out by affinity chromatography on heparin.

3. The method of claim 2, wherein the PLTP is extracted with a salt concentration of greater than 250 mM.

4. The method of claim 1, wherein ultracentrifugation is used to clarify the product in step (iii).

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Comparison of the phospholipid transfer activities in the milks of rabbits WT (Milk A and Milk B) and PLTPTg.sub.WAP (Milk 04, Milk 06 and Milk 13). On the x-axis: time in minutes; on the y-axis: phospholipid transfer activity (in fluorescence units).

(2) FIG. 2: FPLC profile for elution of the proteins of milk clarified on Heparin Sepharose HR26/16 column, using a discontinuous NaCl gradient with 50 mM increases. On the x-axis: elution volume; on the y-axis: absorbance at 280 nm.

(3) FIG. 3: Polyacrylamide gradient gel electrophoresis under denaturing conditions of fractions #167, #175 and #178 eluted on Heparin Sepharose column.

(4) FIG. 4: FPLC profile for elution of the proteins of milk clarified on Heparin Sepharose HR26/16 column, using a discontinuous NaCl gradient with 100 mM increases. On the x-axis: elution volume in milliliters; on the y-axis: absorbance at 280 nm.

(5) FIG. 5: Polyacrylamide gradient gel electrophoresis under denaturing conditions of fraction #68 eluted on Heparin Sepharose column and having a high phospholipid transfer specific activity. unbound: unbound fractions; bound: bound fractions.

(6) FIG. 6: Second electrophoresis run (same as FIG. 5 with a shorter time for staining with Commassie blue). bound: bound fractions.

(7) FIG. 7: Phospholipid transfer activity in plasma from PLTP-KO mice injected either with the purified PLTP fraction (black bars), or with an identical volume of PBS buffer (white bars). On the x-axis: the different sampling times; on the y-axis: phospholipid transfer activity (in fluorescence units).

(8) FIG. 8: Plasma concentrations of cytokines after injection of LPS, in animals having received recombinant PLTP (black bars), or PBS buffer (white bars).

(9) FIG. 9: Area under the curve for the cytokine concentrations measured over a period of 24 hours and expressed in pg/ml of plasmahour.

(10) FIG. 10: Phospholipid transfer activity of the plasma of PLTP-KO mice having received 15 mg/kg of LPS, and injected either with the purified PLTP fraction (black bars), or with an identical volume of PBS buffer (white bars).

(11) FIG. 11: Kaplan-Meier curves showing the mortality of PLTP-KO mice which have received a single dose of LPS (15 mg/kg, i.p.). Arrows: several intravenous injections (caudal vein) of buffer (PBS) or of recombinant PLTP (PLTP) at the times 15 min, 1 h, 2 h, 3 h, 4 h, 5 h and 7 h post LPS.

(12) FIG. 12: Kaplan-Meier curves showing the mortality of PLTP-KO mice which have received a single dose of LPS (25 mg/kg, i.p.). Arrows: a single injection of LPS and then several intravenous injections (caudal vein) of active recombinant PLTP (active PLTP) or inactivated recombinant PLTP (inactivated PLTP) at the times 1 h, 5 h, 10 h, 24 h, 32 h, 48 h, 56 h, 72 h and 80 h post LPS.

(13) FIG. 13: Comparison of the phospholipid transfer activities of active recombinant PLTP, of inactivated recombinant PLTP (2 h, 65 C.), of a wild-type mouse plasma (WT), of a PLTP-KO mouse plasma, and of a PBS buffer. On the x-axis: time in minutes; on the y-axis: phospholipid transfer activity (in fluorescence units).

EXAMPLE 1: CONSTRUCTION OF A TRANSGENESIS VECTOR CONTAINING THE SEQUENCE ENCODING HUMAN PLTP

(14) Cloning of PLTP cDNA into an Intermediate Vector

(15) A backtranslated synthetic cDNA sequence encoding the human PLTP precursor (UniProtKB/Swiss-Prot P55058), bordered, at the 5 position, by a consensus Kozak sequence and by a unique MluI restriction site and, at the 3 position, by two stop codons and by an NheI restriction site, was synthesized and cloned into an intermediate vector, derived from the plasmid pBluescript, containing the ampicillin resistance gene as well as the Col E1 bacterial replication origin.

(16) Transgenesis Vector

(17) The transgenesis vector used for the cloning is derived from the plasmid pPolyIII, having an ampicillin resistance gene as well as the Col E1 bacterial replication origin. This transgenesis vector contains an expression cassette comprising: a dimer of the 5HS4 insulator sequence of the chicken beta-globin gene (Genbank U78775) (RECILLAS-TARGA et al., Proc. Natl. Acad. Sci., 10: 6883-6888, 2002) upstream of the 6.3 kbp rabbit WAP promoter (whey acidic protein; Genbank X52564) (RIVAL-GERVIER et al., Transgenic Res., 6: 723-730, 2003), the second intron of the rabbit beta-globin gene (Genbank V00882) containing a transcription enhancer; a second transcription enhancer (SUR 1.2.3) containing the 5UTR sequence of the SV40 early genes, fused with the R region and the start of the HTLV-1 U5 region (ATTAL et al., FEBS Lett., 392, 220-224, 1996); a third transcription enhancer (Ig2), derived from the mu region of the mouse IgG heavy chain (Genbank J00440) (GILLIES et al., Cell 33, 717-728, 1983) and the transcription terminator of the human growth hormone (Genbank M13438). This cassette contains an MluI site and an NheI site located between the second and third transcription enhancer; it is flanked on either side by NotI sites allowing the excision of sequences of the plasmid pPolylII.

(18) The DNA insert encoding PLTP, recovered by MlullNheI digestion from the intermediate vector, was inserted between the MluI and NheI sites of the expression cassette.

(19) The resulting transgenesis vector contains an insert which consists, in its 5 to 3 orientation, of i) the dimer of the 5HS4 insulator sequence of the chicken beta-globin gene, ii) the 6.3 kbp rabbit WAP promoter (whey acidic protein), iii) the intron containing the first transcription enhancer, iv) the second transcription enhancer, v) the human PLTP cDNA, yl) the third transcription enhancer, and vii) the transcription terminator.

(20) As above, the colonies containing the recombinant vector are selected on the basis of their ampicillin resistance, and then the presence of the insert is checked by analysis of the restriction fragments, and then by sequencing.

EXAMPLE 2: PRODUCTION OF TRANSGENIC RABBITS EXPRESSING THE HUMAN PLTP IN THEIR MAMMARY GLANDS

(21) The transgenic rabbits were obtained by the conventional microinjection technique (BRINSTER et al., Proc. Natl. Acad. Sci., 82: 4438-4442, 1985).

(22) Preparation of the Inserts for Transgenesis

(23) The transgenesis vector containing the sequence encoding recombinant PLTP was digested by the restriction enzyme NotI and the insert containing the transgene was isolated on agarose gel and then purified on ElutipD (Schleicher-Schuell, Ecquevilly, France) in accordance with the manufacturer's instructions, precipitated with ethanol and then taken up in a buffer containing 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4.

(24) Preparation of the Donor and Recipient Rabbits

(25) Embryo-donating New Zealand rabbits, aged 16-30 weeks, are treated by the subcutaneous route for 3 days with porcine FSH (Follicle Stimulating Hormone) in order to stimulate follicle development. On the third day, the rabbits receive an intravenous injection of hCG hormone (human Choroidic Hormone), and then the females are mated.

(26) The recipient rabbits are aged 18-20 weeks. A synchronized pseudogestation is induced either by keeping the females for one week in a long day cycle (16 h of light) before they are mated with the vasectomized males, or by a hormone treatment (FSH/LH superovulation).

(27) Microinjection of Embryos Collected and Implantation

(28) On the 4th day, 18-19 h after mating, the embryos are collected from the donor rabbits in order to carry out the microinjection of DNA: the microinjection of DNA is carried out immediately after the collections (15-25 h after mating). The embryos at the one-cell stage are placed in a microdrop of medium under an inverted microscope equipped with Normarsky lenses and with micromanipulators. Individual embryos are positioned and secured using a pipette. The transgene, diluted at a concentration which may vary from 1 ng/l to 6 ng/l, in buffer containing 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4, is preferably microinjected into the male pronucleus of the embryo with the aid of a pipette for injection.

(29) Following the microinjection, the embryos are maintained in culture in vitro for 1 to 5 h (35 to 40 C., 3 to 5% CO.sub.2 in air). The quality of the microinjected embryos is then rapidly evaluated under a stereomicroscope. The intact unicellular embryos are then reimplanted under general anesthetic into the lumen of the oviducts of the synchronized recipient rabbits (10 embryos into each oviduct), using a surgical procedure (the oviducts are exteriorized by laparotomy). Parturition may occur naturally 29-31 days after the transfer of embryos. If necessary, it is triggered by injecting ocytocin on the 31st day. The number of young rabbits born compared with the number of embryos reimplanted is of the order of 5 to 20%.

EXAMPLE 3: SELECTION AND CHARACTERIZATION OF THE TRANSGENIC RABBITS

(30) During the embryogenesis process, the microinjected recombinant DNA is randomly integrated into the genome. The newborn rabbits (10 days) are tested for the presence of the transgene by a biopsy of the ear. The genomic DNA is extracted and PCR (Polymerase Chain Reaction) analysis is carried out using primers specific for the recombinant insert.

(31) The rabbits for which the transgene was detected are called Founders F0.

(32) The founder F0 lines were in addition characterized by i) analysis of the number of copies of transgene integrated into their genome, and ii) determination of the number of sites of integration.

(33) The number of copies of the transgene integrated into the genome of each founder F0 line was determined by quantitative PCR and by Southern blotting. This number varies, depending on the line, from 1 copy per cell to about one hundred copies per cell.

(34) The number of sites of integration was also determined by Southern blotting. This number varies, depending on the line, from 1 to 3 sites per genome.

EXAMPLE 4: EVALUATION OF THE EXPRESSION OF HUMAN RECOMBINANT PLTP IN THE MILK OF THE TRANSGENIC RABBITS

(35) The phospholipid transfer activity in the milk of F0 transgenic rabbits of Example 3 was evaluated.

(36) For that, the phospholipid transfer activity by PLTP was measured in the milk samples using the commercial kit from Roar Biomedical Inc. (New York, N.Y., USA) according to the recommendations of the manufacturer. A fluorescent phospholipid, in this case phosphatidylcholine, is present in an auto-extinction state when it is combined with a liposome-type donor substrate. The phospholipid transfer activity of PLTP is determined by increasing the fluorescence intensity when the fluorescent phospholipid is transferred to an acceptor lipoprotein substrate, for example of the VLDL type (very low density lipoprotein), present in the incubation medium (Masson D. et al. Mol Human Reprod 2003; 9: 457).

(37) FIG. 1 represents the comparison between the phospholipid transfer activity in the milk of non-transgenic rabbits, or WT rabbits (Milk A and Milk B), and the milk of several lines of rabbits transgenic for PLTP, or PLTPTg.sub.WAP rabbits (Milk 04, Milk 06 and Milk 13). When no significant phospholipid transfer activity is detected in the milk of WT rabbits, the milk of PLTPTg.sub.WAP rabbits show a time-dependent capacity to exchange fluorescent phospholipids between synthetic liposomes and plasma VLDL lipoproteins. Milk 13, which exhibits the highest phospholipid transfer activity, was used for the purification of the protein.

EXAMPLE 5: PRODUCTION OF AN ACTIVE HUMAN PLTP PREPARATION FROM TRANSGENIC RABBIT MILK

(38) 1Precipitation of Caseins at Acidic pH and Room Temperature

(39) 1 volume of milk is mixed with 2 volumes of 0.5 mM EDTA, pH 8.0 and 7 volumes of MilliQ water. The EDTA makes it possible to chelate the calcium ions and to break the casein micelles which are capable of retaining part of the PLTP.

(40) The pH is gradually reduced to 4 by adding glacial acetic acid dropwise with stirring, in order to completely precipitate the caseins (whose pH, is 4.6).

(41) 2Clarification of the Whey by Low-Speed Centrifugation and Neutralization of the pH

(42) After precipitation of the caseins at acidic pH, centrifugation of the mixture for 10 min at 2000 g and at 4 C.

(43) Collection of the intermediate clarified fraction situated between the lipid supernatant and the protein pellet.

(44) Neutralization with solid Tris in a sufficient quantity for obtaining pH 7.4.

(45) Filtration of the fraction thus collected on a glass fiber filter (Millipore AP2004700). It is a conventional filtration, before injection onto the column. It makes it possible to remove the aggregates and/or particles of large size and to thereby protect the column by avoiding its blockage by these aggregates and/or particles of large size.

(46) 3Extraction/Purification of PLTP by Affinity Chromatography on a Heparin Sepharose Column

(47) Injection of the clarified and filtered protein fraction, at room temperature, onto a Heparin Sepharose 6 Fast Flow column (24016 mm ID) equilibrated beforehand with a 20 mM Tris buffer, pH 7.4.

(48) Rate of injection: 1 ml/min with a peristaltic pump.

(49) Rinsing of the column overnight with a 20 mM Tris buffer, pH 7.4.

(50) Connecting of the loaded column to an Akta FPLC system.

(51) Programming of a discontinuous gradient of the 20 mM Tris buffer, pH 7.4 to the 20 mM Tris/1 M NaCl buffer, pH 7.4 (with increases of 50 or 100 mM NaCl).

(52) Dialysis of the fraction against a 150 mmol PBS/1 NaCl buffer, pH 7.4.

(53) The phospholipid transfer activity is measured in each of the fractions (according to the same protocol as in Example 4), and compared with the values for BCA protein assay (assay with a commercial Bicinchoninic Acid Assay kit, Interchim, Montluon, France).

(54) Results

(55) The results of an extraction carried out using a discontinuous NaCl gradient with 50 mM increases are illustrated by FIGS. 2 and 3.

(56) As shown in FIG. 2, the proteins of the skimmed fraction of milk are eluted from the heparin column in several successive peaks according to the discontinuous NaCl gradient. Several discrete peaks (fractions #167, #175 and #178) are eluted at the highest NaCl concentrations (greater than 250 mM).

(57) The results of the measurement of phospholipid transfer activity are summarized in Table I below.

(58) TABLE-US-00001 TABLE I SA fraction/SA Specific milk Tg [prot] g/l Activity activity (SA) ratio Milk Tg 92.73 17781 192 1 Fraction #61 0.185 47 254 1 Fraction #91 0.163 379 2325 12 Fraction #102 0.185 496 2681 14 Fraction #119 12.398 4119 332 2 Fraction #149 5.44 5750 1057 6 Fraction #167 0.215 3262 15172 79 Fraction #175 0.21 2256 10743 56 Fraction #178 0.469 4715 10053 52

(59) The specific activity for phospholipid transfer (Specific activity (SA) column of Table I) is highest in fractions #167, #175 and #178, where it is more than 50 times higher than in the starting milk (last column on the right of Table I).

(60) FIG. 3 shows that these fractions consist of 2 predominant proteins having an apparent molecular weight of 30 and 60 kDa respectively. The 30 kDa band corresponds to caseins, found in abundance in the WT and PLTPTg.sub.WAP milks. The 60 kDa band is in the region of the MWs conventionally reported for PLTP (between 50 and 70 kDa).

(61) The results of an extraction carried out using a discontinuous NaCl gradient with 100 mM increases are illustrated by FIGS. 4 to 6, and Table II below.

(62) The fraction exhibiting the highest phospholipid transfer specific activity (fraction #68) is eluted at the 300 mM NaCl step (FIG. 4).

(63) This fraction has a specific activity that is 44 times higher than the starting milk (last column on the right of Table II below).

(64) TABLE-US-00002 TABLE II SA fraction/SA Specific milk Tg [prot] g/l Activity activity (SA) ratio Milk WT 126 1193 9 Milk Tg 92.73 12946 140 1 Fraction #2 2.47 3720 1506 11 Fraction #4 14.31 6474 452 3 Fraction #32 0.86 11 13 0 Fraction #44 2.03 4522 2228 16 Fraction #48 1.51 4220 2795 20 Fraction #53 3.67 5411 1474 11 Fraction #68 0.425 2586 6085 44

(65) Electrophoretic analysis under denaturing conditions again shows a major band at 60 kDa corresponding to PLTP, and a more discrete band at 30 kDa corresponding to the caseins (FIG. 5). A second electrophoresis with a less intense staining of the bands shows a single band of 60 kDa for the most active fraction #68 (FIG. 6). These results show that human PLTP is the major constituent of the fraction exhibiting the highest phospholipid transfer specific activity.

EXAMPLE 6: USE OF HUMAN PLTP OBTAINED FROM THE MILK OF TRANSGENIC RABBITS IN THE PREVENTION OF ENDOTOXIN SHOCK IN PLTP-KNOCK-OUT MICE

(66) Previous data (GAUTIER et al., J Biol Chem, 283, 18702-10, 2008) have shown that disabling the endogenous gene for PLTP in mice (homozygous PLTP-KO mice) leads to a reduction in the capacity for neutralizing and detoxifying the LPSs compared to the wild-type WT mice. The mortality after injection of a lethal dose of LPS occurs earlier in the PLTP-KO mice than in the WT mice.

(67) In order to determine if the exogenous supply of recombinant PLTP can increase detoxification of the LPSs and the resistance to endotoxin shock in animals, PLTP-KO mice received, by the intraperitoneal route, a lethal (15 mg/kg) or sublethal (5 mg/kg) injection of LPS at time 0, followed by successive intravenous injections of PBS buffer or of recombinant PLTP (100 microliters of active fraction #68) prepared from the milk of transgenic rabbits as described in Example 5 above. The plasma was then collected by retroorbital puncture in order to assay the circulating concentrations of cytokines (IL-6, IL-10, MCP-1, IFNgamma, TNFalpha), and the phospholipid transfer PLTP activity, according to the protocols described by GAUTIER et al. (2008, cited above). Finally, the mortality rates were recorded over a period of 4 days following the initial injection of LPS.

(68) FIG. 7 illustrates the plasma phospholipid transfer activity in mice, after injection(s) of recombinant PLTP or of PBS buffer. This plasma phospholipid transfer activity is significantly higher 30 minutes after injection of recombinant PLTP compared with mice receiving the PBS buffer. The increase in activity disappears 90 minutes after the first injection of PLTP, but remains significant 60 minutes after the third injection which took place at time 4 hours. Likewise, at time t=9 h, the plasma phospholipid transfer activity is significantly higher in the animals receiving recombinant PLTP compared with the animals receiving the PBS buffer (that is 60 minutes after the last injection of exogenous PLTP at time t=8 h). At time t=24 hours, the animals which received the last injection of recombinant PLTP at time t=8 h no longer exhibit an increase in plasma phospholipid transfer activity compared with the animals which received the PBS buffer.

(69) These results show that the injection of exogenous PLTP in PLTP-KO mice leads to a significant and transient rise in the plasma phospholipid transfer activity with a persistence of the order of 60 minutes.

(70) To study the effects of recombinant PLTP on the production of the cytokines IL-6, IL-10, MCP-1, IFNgamma and TNFalpha, animals having received a single intraperitoneal injection of LPS at sublethal dose (5 mg/kg) then received several intravenous injections (caudal vein) of PBS buffer or of recombinant PLTP, 10 min, 2 h, 4 h, 6 h and 8 h after the administration of the LPSs. Retroorbital blood samples were collected at times T 0, T 30 min, T 1 h 30 min, T 5 h, T 9 h and T 24 h in the animals anesthetized with isoflurane, and the cytokines were assayed.

(71) The results are illustrated by FIGS. 8 and 9.

(72) After injection of the single dose of LPS at 5 mg/kg, the circulating concentrations of cytokines follow a biphasic variation during the 24 hour period studied, with an initial phase of increase, followed by a phase of decrease leading at t=24 h to the background values measured in the PLTP-KO mice at time t=0 (FIG. 8). A significant reduction in the circulating concentrations of IL-6, IL-10 and IFNgamma is observed at various times in the mice receiving recombinant PLTP compared with the mice receiving PBS. The cytokines IL-6, IL-10, MCP-1, IFN and TNF show biphasic curves, with significantly lower IL-6 and IFN production. The calculation of the area under the curve (AUC) during the 24 hour period studied shows significantly lower values of IL-6 (p=0.058) and of IFNgamma (p=0.036) in mice receiving the recombinant PLTP by the intravenous route than in those which received PBS (FIG. 9).

(73) In order to study the effect of exogenous PLTP on mortality, the intraperitoneal injection of a single lethal dose of LPS at 15 mg/kg was followed by a sequence of intravenous injections of exogenous PLTP at times t=15 min, t=1 h, t=2 h, t=3 h, t=4 h, t=5 h, t=7 h after injection of the LPSs. Retroorbital blood samples were collected in the animals anesthetized at times T 0, T 1 h 30 min and T 6 h after injection of the LPSs, and the plasma phospholipid transfer activity was determined. The length of survival of the animals was noted in parallel.

(74) The results are illustrated in FIGS. 10 and 11.

(75) As shown in FIG. 10, the plasma phospholipid transfer activity is significantly higher 30 minutes after the second injection in the mice receiving recombinant PLTP compared with the mice receiving the PBS buffer. An increase in activity (of the order of 5 times) is observed at time t=6 h, that is 1 hour after the 4.sup.th injection of recombinant PLTP. Again, these results show that the cumulative injection of exogenous PLTP in the PLTP-KO mice leads to a significant and transient rise in the plasma phospholipid transfer activity with a persistence of at least 60 minutes. The Kaplan-Meier curves (FIG. 11) show the extension of the survival of the animals which received the exogenous recombinant PLTP compared with the animals which received the PBS buffer. The average lengths of survival are 33.65.4 h (meansem) for the mice which received PBS (n=8) and 77.16.2 h (meansem) for the mice (n=7) which received the PLTP (p<0.05 against PBS by the Chi2 test).

(76) These results demonstrate the beneficial effect of an exogenous PLTP supply in animals subjected to an endotoxin shock by a single intraperitoneal injection of LPS. Successive injections of PLTP during the first few hours following the injection of LPS significantly reduce the production of inflammatory cytokines and extend survival.

EXAMPLE 7: USE OF ACTIVE OR INACTIVATED HUMAN PLTP OBTAINED FROM THE MILK OF TRANSGENIC RABBITS IN THE PREVENTION OF ENDOTOXIN SHOCK IN PLTP-KNOCK-OUT MICE

(77) In order to confirm the effect of exogenous PLTP on the mortality, observed in Example 6, the intraperitoneal injection of a single lethal dose of LPS (25 mg/kg of body weight) was followed by a sequence of intravenous injections of recombinant PLTP (200 microliters, 2.2 g proteins/1), prepared from the milk of transgenic rabbits as described in Example 5, at times t=1 h, 5 h, 10 h, 24 h, 32 h, 48 h, 56 h, 72 h and 80 h after the injection of LPS.

(78) The survival rate was followed as a function of time, as represented in FIG. 12. The effect of the active recombinant PLTP was compared with that of a PLTP inactivated by heating a preparation of recombinant PLTP, purified according to Example 5, for 2 hours at 65 C. The Kaplan-Meier curves were compared by the Chi2 test. After 4 days, all the mice injected with LPS and then inactivated PLTP died. The injection of active recombinant PLTP makes it possible to very significantly increase (p<0.05 against inactivated PLTP) the survival of the animals. More than 60% of the mice are permanently saved (beyond 7 days). The intravenous injection of active exogenous recombinant PLTP, as opposed to the same PLTP inactivated by heating, therefore increases the survival of the PLTP-KO mice which received an injection of a lethal dose of LPS.

(79) The phospholipid transfer activity of the inactivated PLTP was measured by fluorescence with the aid of a commercial kit (Roar Biomedical Inc.), as in Example 4, and compared with those of a PBS buffer, of active recombinant PLTP, of a wild-type (WT) mouse plasma, and of a PLTP-KO mouse plasma. The results, illustrated by FIG. 13, show that only the WT mouse plasma and the active recombinant PLTP make it possible to detect a phospholipid transfer activity, the inactivated recombinant PLTP, like the PBS buffer and the PLTP-KO mouse plasma, resulting in no significant activity.

(80) These results confirm the beneficial effect of an exogenous PLTP supply in animals subjected to an endotoxin shock by single intraperitoneal injection of LPS. Successive injections of PLTP during the first few hours following the injection of LPS significantly extend survival.