FR2796385A1 - METHOD FOR PRODUCING ANTIBODY IN EGG YELLOW AND USE OF THE ANTIBODIES THUS OBTAINED - Google Patents

Abstract

The invention concerns a method for producing antibodies in egg yolk comprising a step which consists in immunising aviaries with a naked DNA coding for a protein antigen, collecting the eggs and optionally purifying the antibodies produced in the egg yolks. The invention also concerns the use of the resulting antibodies for human or veterinary immunotherapy and for diagnosis.

Description

The present invention relates to a method for producing antibodies in egg yolk and the use of antibodies thus obtained in human or veterinary immunotherapy and as a diagnostic tool.

F. Klemperer observed for the first time that immunized hens transmit serum immunoglobulins (1-G) to egg yolk (Arch Erp Pathol Pharnaacol (1893), 31, 356-382). The yolk antibodies, called IY, are considered to be analogs of human IG but have a very different structure, especially at the level of the heavy chain (G. Camenish. <I> et al. </ I> <I the FASEB Journal, (l999) 13, 81-88).

The production of antibodies in egg yolk, after immunization with an antigen, has several advantages, in particular - the amount of antibody per yolk is of the order of 10 mg / g whereas it is 35 mg / ml in rabbit serum; knowing that a hen lays about 250 eggs per year, then after immunization at 0, 2, 4, 6, 22 and 28 weeks, a total of 40 g of antibody is recovered, whereas in the rabbit only 1.4 g are recovered. after bleeding from the animal. Thus the amount of antibody produced per hen is about 30 times higher than that of the antibodies contained in the serum of a rabbit, (Natta H. Biosci, Biotech Biocheni., (1993) 57, 450-454), antibodies contained in egg yolk which do not exhibit cross-reactions with mammalian IgGs, because of their structural differences (Natta H., (reference cited), Liu SS Comp Bioches Physiol (l999). 97B, 637-644), making it a reagent of choice in immunodiagnostic tests, a non-invasive means of harvesting eggs, and simple methods for isolating antibodies (Natta H. cited reference).

However, this technique is limited by the fact that it requires the expression and purification of the recombinant proteins used as immunogens. Indeed, the expression of recombinant proteins is one of the limiting factors for the production of specific antibodies, because some proteins are poorly expressed, or expressed in a conformational form that is different from the native protein or expressed in a specific form. insoluble form that can not be used as an immunogen. The existence of many variants limits the use of these antibodies as a diagnostic tool.

Immunization with naked DNA, on the other hand, does not require protein expression or purification, which is sometimes expensive and very difficult to perform. It makes it possible to produce antibodies which have a protective effect (Tang DC <I> et al., Nature, </ I> (1992), 356, 152-154) and induces a cellular immune response characterized by the production of T lymphocytes, capable of recognizing and lysing infected cells (Ulmer JB <I> et al. </ I> ASM <I> News </ I> (1996), 62, 476-479).

Using this method, which has shown great promise in several animal models of infections caused by vinas, bacteria, parasites or mycoplasmas (Lai WC, <i> et al. </ I> Critical Revieves <I > In </ I> Iinnaunology (1998), 18, 449-484), the inventors have recently shown that the immunization of ducks, by a vector expressing the envelope protein, protein L, of the hepatitis B virus duck (DHBV), induces a strong, specific and long-lasting humoral response that is able to neutralize the virus and protect newborn ducklings against viral infection (Rollier C. <I> et al. </ I Gastroenterology (1999), 116, 658-665).

However, this method is not suitable for producing antibodies on an industrial scale and the antibodies obtained have cross-reactions which make them unusable in humans in immunotherapy.

Also, continuing their work, the inventors set themselves the goal of providing a method for the production of antibodies that overcomes the drawbacks of existing methods, that is to say, a method that is simple to implement, fast, with little expensive, and which allows to produce, in large quantities, specific antibodies, which can be used in human or veterinary immunotherapy, and also as a diagnostic tool.

The subject of the present invention is therefore a method of producing antibodies in egg yolk, characterized in that it comprises - the immunization of avians with a naked DNA, - the collection of the new ones and then the yeasts. eggs, and - possibly the purification of the antibodies produced in the egg yolks.

In a particular embodiment of said method, the immunization is carried out in the hen or cane.

For the purposes of the present invention, the term "naked DNA" means both a DNA encoding an appropriate antigen or protein and cloned into a vector, in particular into a plasmid, than linear DNA fragments which express a eukaryotic gene or prokaryote, especially the Linear Expression Elements (LEEs) such as those described by Sykes KF <I> et al. (Nature </ I> Biotechnologv (l999), 17, 355-359). For the implementation of the method according to the invention, the DNA can be injected either intradermally, intramuscularly or intravenously, orally or intramuconasally.

The DNA may be injected in any form known to those skilled in the art, especially in saline solution, in the form of complexes with lipids, in the form of an aerosol, adsorbed on gold beads or encapsulated in microparticles.

For the purposes of the present invention, the DNA used encodes an antigen of interest, in particular for eukaryotic or prokaryotic proteins. Among the DNAs encoding eukaryotic proteins, there may be mentioned the DNAs encoding antigens expressed by tumor cells, insulin, allergenic agents, toxins, venoms and growth factors.

 Among the DNAs encoding prokaryotic proteins, mention may be made of the DNAs encoding viral proteins, in particular the proteins of HIV (human immunodeficiency virus), influenza, rabies virus and rotaviruses. , hepatitis B, C, D and E, human cytomegalovinis, herpes viruses, dengue virus, polio virus, measles virus, HTLV I (Human T-cell Leukemia Virus ), NDV <I> (Newcastle </ I> Disease <I> Virus), </ I> Ebola virus, papillomavirus and Sendai virus, </ I> - proteins of bacterial origin , in particular the proteins of Mycobacterium tuberculosis and Bovis, of Salmonella type, of Clostridium telarni, of schistosome, and of the plasmoplasma, the protozoan proteins, in particular the proteins of Plasmodium. / I> Yolli (Malaria Agent) or Leishmania or Toxoplasnaa Gardii. The method according to the invention makes it possible to rapidly prepare, in large amounts, antibodies specific for different antigens and their variants which at present can not be detected.

The present invention also relates to the use of at least one antibody obtained by the method according to the invention, said antibody being optionally combined with a suitable substance, for the preparation of an immunological reagent that can be used for screening, diagnosis and / or monitoring of diseases in humans and animals.

Among the said diseases, there may be mentioned infectious diseases, cancers and allergies. The present invention also relates to a method for detecting, diagnosing and / or monitoring a disease, in humans or in animals, comprising detecting the presence of an antigen possibly present in a biological sample, especially in the blood, characterized in that it uses at least one appropriate antibody produced in accordance with the invention.

In the context of said method, when the antigen is present in the biological sample to be tested, it then binds to the antibody and said binding is revealed by any appropriate means known to those skilled in the art, in particular by RIA, EIA, ELISA, or by immunofluorescence and immunoelectromieroscopy techniques.

The present invention also relates to a diagnostic kit or kit for the detection, diagnosis and / or monitoring of a disease, characterized in that it uses at least one antibody obtained according to the method according to the invention, suitable reagents and means for detecting formation of the antigen-antibody bond.

The present invention also relates to the use of egg yolks, or egg yolk fractions containing the antibodies produced according to the invention or purified antibodies produced according to the invention, for the preparation of a drug.

In a particular embodiment of this use, the drug is useful in immunotherapy.

In an even more particular embodiment of said use, the medicament is intended to be administered orally, optionally as a food additive, for passive immunization against enteric pathogenic viruses, in particular rotaviruses, or against oral infections.

In yet another particular embodiment of said use, the medicament is for treating an infection selected from the group consisting of infections of viral origin, bacterial and those due to protozoa.

deuxième cane, (-O-) troisième cane. In another embodiment of said use, the medicament is intended to treat a disease selected from cancers and allergies. Other features and advantages of the invention appear in the following description and examples illustrated by the figures in which - Figure 1 illustrates the kinetics of the humoral response in 3 channels, after immunization with the plasmid pCI-preS / S according to the method described in Example 1. The detection of the anti-preS antibodies was carried out by a direct ELISA test according to the procedure described in Example 1. The arrows indicate the DNA injections carried out 4, 7, 10 and 28 weeks after birth (-x first cane,

second cane, (-O-) third cane.

Les résultats sont exprimés en moyenne s.e.m. (n=3). La détection des anticorps anti-preS a été réalisée par un test ELISA direct, selon le mode opératoire décrit dans l'exemple 1. La flèche indique l'injection d'ADN. FIG. 2 illustrates the evolution of the average anti-preS antibody titre in the egg yolk of canes immunized with the empty plasmid pCI according to the method described in Example 1 (-O-), or in the serum of canes immunized with the plasmid pCI-preS / S according to the method described in Example 1 (-x), or in yolk of cane eggs immunized with the plasmid pCI-preS / S according to the method described in example 1

The results are expressed on average sem (n = 3). The detection of the anti-preS antibodies was carried out by a direct ELISA test, according to the procedure described in Example 1. The arrow indicates the injection of DNA.

FIG. 3 illustrates the specificity of the purified antibodies (IgY) obtained according to the method according to the invention. Serum proteins infected with DHBV (DHBV +) and uninfected serum (DHBV -) were revealed by immunoblotting with (A) a polyclonal rabbit anti-DHBpreS antiserum, (B) a pCI-immunized serum serum. preS / S, (C) eggs of eggs laid by pCI-preS / S immunized eggs and (D) IgY of eggs laid by canes immunized with the empty plasmid pCI. p36 corresponds to the L protein of DHBV.

pré-incubation avec du tampon phosphate,

pré-incubation avec du sérum de cane témoin immunisée avec le plasmide vide pCI, (- + ) pré-incubation avec des I-Y témoins,

pré-incubation avec du sérum de canes immunisées par le plasmide pCI-preS/S, (--x--) pré-incubation avec des IgY d'oeufs de canes immunisées par le plasmide pCI preS/S. EXEMPLE 1 : Production d'anticorps dans le jaune d'oeuf après immunisation par ADN 1.1<U>Matériel et méthodes</U> l.1.1. Préparation_du_plasmide Le fragment d'ADN du DHBV codant pour le gène preS/S (nucléotides 801-178S) est cloné par PCR dans le site Not <I>I</I> du site multiple de liaison (polylinker) du vecteur pCI (Promega, Charbonnières, France) permettant d'obtenir le plasmide pCI-preS/S. Ce plasmide pCI-preS/S et le plasmide pCI (témoin négatif) sont introduits dans Escherichia colï et les ADN sont purifiés par chromatographie échangeuse d'anions sur des colonnes Qiagen (Qiagen, Hilden, Germany) et quantifiés par mesure de la densité optique à 260 nm avec marquage au bromure d'éthidium. FIG. 4 illustrates the in vitro neutralization of the infectivity of DHBV by canine serum or by antibodies (IgY) of cane egg yolks. Primary cultures of canine hepatocytes are infected with an inoculum of DHBV pre-incubated with serum or IY according to the procedure described in Example 2. The release of the viral particles is quantified in the culture medium by a method molecular quantitative hybridization <I> (dot </ I> blot hybridization). The results are expressed as mean sem (n = 2).

preincubation with phosphate buffer,

pre-incubation with immune control cane serum with the empty plasmid pCI, (- +) pre-incubation with control IY,

pre-incubation with serum of canes immunized with the plasmid pCI-preS / S, (- x--) pre-incubation with IgY of cane eggs immunized with plasmid pCI preS / S. EXAMPLE 1 Production of Antibodies in Yolk After Immunization with DNA 1.1 <U> Materials and Methods </ U> l.1.1. Preparation of the plasmid The DHBV DNA fragment encoding the preS / S gene (nucleotides 801-178S) is cloned by PCR into the NotI I site of the polylinker of the pCI vector (Promega , Charbonnières, France) making it possible to obtain the plasmid pCI-preS / S. This plasmid pCI-preS / S and the plasmid pCI (negative control) are introduced into Escherichia coli and the DNAs are purified by anion exchange chromatography on Qiagen columns (Qiagen, Hilden, Germany) and quantified by measuring the optical density. at 260 nm with ethidium bromide labeling.

1.1.2. Channel immunization One group of canes is immunized by intramuscular injection into the right anterior quadriceps, 100 μg of plasmid pCI-preS / S coding for DHBV L protein, in solution in 0.9% NaCl, another group receiving under the same conditions the plasmid pCI.

The DNA injections are performed at weeks 4, 7, 10 and 28 after birth, at a rate of <B> 100 </ B> #tg of DNA per injection for weeks 4.7 and 10 and 200 # tg of DNA per injection for week 28.

1.1.3. Antibody Purification Antibodies are extracted and purified from each egg yolk using the EGGstract kit (Promega) following the supplier's instructions.

1.1.4. Assay of Antibody by ELISA Anti-preS antibodies are detected by a direct ELISA using DHBpreS polypeptide coated microtiter plates, blocked, washed and revealed by the method described by S. Chassot et al. (1994), 200, 72-78). The final titer is considered to be the inverse of the highest serum dilution q u <B> 1 </ B> gives a higher optical density <B> than </ B> SD above the average signal of the controls.

1.2 <U> Results </ U> 1.2.1. Antibody production in serum: The results are illustrated in Figure 1.

The kinetics of the humoral response, in the serum of three canes, shows that the immunization with naked DNA induces a high level of anti-preS antibodies in the serum of the order of 3200 to 14,000 on average by blood sample. .

The amount of antibody reaches its maximum from the first DNA injection, then a plateau is observed which is maintained until 40 weeks after birth.

On the other hand, in canes immunized with the plasmid pCI, the production of antibodies (data not shown) is not observed. 1.2.2. Antibody Product in Yolk The results are shown in Figure 2.

The antibody titre per oruf is 3200 to 6400 for all widowers collected during the six weeks.

The amount of purified antibody per egg harvested is about 60 to 100 mg and without significant variation from one egg to another.

During the 6 consecutive weeks of the experiment, no decrease in the titre of antibodies, either in the serum or in the yolks of eggs laid in the weeks following the DNA injection, is observed and this, even in the absence of a new DNA injection.

When a new one is collected per week per animal, the total amount of antibodies produced during the 6 weeks is 360 to 600 mg per animal, and this is non-invasive.

Knowing that one cane lays one egg per day, when all the widows laid by an animal are harvested, the total amount of antibodies produced during the 6 weeks is 2.5 to 4.2 g per animal.

No antibody production is detected in the non-immunized egg yolks.

EXAMPLE 2 Specificity of the Antibodies Present in the Yolks of Eggs

2.1 <U> Materials and methods </ U> 2.l.1. Immunoblotting ------------------ The particles of DHBV are prepared by ultracentrifugation of serum of canes with a high viremia and the particles serving as negative controls are prepared by ultracentrifugation of serum of canes. uninfected.

The liver proteins of canes infected or not with DHBV are extracted according to the technique described by S. Chassot <I> et al. </ I> (Virolop: (1993), 192, 217 223).

The method is that described by Borel C.I. et al. (Virology (1998), 242, 90-98) and uses either a polyclonal anti-DHBpreS rabbit antiserum (1: 1000), or a serum of pCI-preS / S immunized canines, either eggs of eggs laid by pCI-preS / S immunized eggs, or IgG of eggs laid by pIC-immunized canes. After washing, the filters are incubated with peroxidase G conjugates and anti-rabbit, anti-mouse or anti-duck immunoglobulins and the reaction is measured by chemiluminescence (Amersham kit, Courtaboeuf, Les Ullis France). 2.2 <U> Results </ U> They are illustrated in Figure 3.

The IgY antibodies produced in the egg yolks after immunization with pCI-preS / S recognize the DHBV envelope protein, protein L, with a molecular weight of 36 kDa. present in concentrated particles derived from serum (Figure 3C).

The immunoblotting profile is comparable to that obtained with the serum of canes previously immunized with the plasmid pCI-preS / S (FIG. 3B), or with the rabbit serum immunized with the recombinant envelope protein of DHBV (FIG. 3A). .

After immunization with the plasmid pCI, no reactivity was observed with the DHBV envelope protein (FIG. 3D).

EXAMPLE 3 In Vitro Measurement of the Neutralizing Potency of Antibodies Produced in Egg Yolks 3.1. <U> Materials and Methods </ U> 3.1.1. Virus The viral inoculum is prepared from a pool of duckweed serum that has been transfected with DHBV cloned and sequenced by Mandart E. <i> et al. </ I> (J. Virol. (1984), 49, 782-792) and quantified in virus genome equivalents (vge) by quantitative molecular hybridization <I> (dot </ I> blot hybridization) as described by S. Borel (1998) (reference cited).

3.l.2. <U> Measurement </ U> of neutralizing capacity <I> in vitro </ I> <B> --------------------- ----------------- </ B> The neutralization test is carried out according to the method described by Rollier C. et al. (1999) (cited reference).

Primary cultures of duck hepatocytes, obtained by perfusion of the liver, in two stages, with collagenase, according to the technique described by C. Borel <I> et al. </ I> (l998) (reference cited), are infected with DHBV-positive serum (2 × 10 6 vge / well) previously incubated overnight at room temperature, either in the presence of serum of immunized canes (1: 10), or in the presence of phosphate buffer.

The release of virus particles is monitored for 8 days by quantitative molecular hybridization.

The area under the curve of DHBV levels in supernatants reflects total viral secretion. Neutralization is effective when the decrease in total viral secretion is at least 50%.

3.2 <U> Results </ U> They are illustrated in Figure 4.

In primary cultures of duck hepatocytes infected with inoculum of DHBV pre-incubated with phosphate buffer, a significant amount of virus released is observed.

Pre-incubation of the inoculum with control cell serum or with yolk egg yolk IgI immunized with the plasmid pCI, does not modify the viral secretion.

On the other hand, after a pre-incubation of the inoculum with serum of canes immunized with the plasmid pCI-preS / S, or with IgY of egg yolk eggs immunized with the plasmid pCI-preS / S, respectively, a 96% and 99% decrease in the release of virions in the medium.

EXAMPLE 4 In Vivo Measurement of the Protective Power of the Antibodies Produced in Egg Yolks 4.1 Material and Methods Infected eggs of canes immunized either with the empty plasmid pCI either by the plasmid pCI-preS / S are incubated until hatching.

Two days after birth, the ducklings are inoculated with DHBV and the humoral response and viremia are measured for 17 days.

4.2 <U> Results </ U> They are illustrated in Table I which follows

Table <SEP> 1
<tb><SEP> Immunization <SEP> N <SEP><SEP> Duckling <SEP> Title <SEP> in <SEP> Antibody <SEP> to <SEP> Viremia <SEP> (10 '<SEP> vge / ml)
<tb> canes <SEP> the <SEP> birth
<tb> pCI <SEP> 927 <SEP> 0 <SEP> 118
<tb> 928 <SEP> 0 <SEP> 5468
<tb> 929. <SEP> 0 <SEP> 191
<tb> 930 <SEP> 0 <SEP> 7726
<tb> 931 <SEP> 0 <SEP> 335
<tb> 932 <SEP> 0 <SEP> 2361
<tb> pCI-preS / S <SEP> 772 <SEP> 12800 <SEP> 0

773 <SEP> 6400 <SEP> 0
<tb> 777 <SEP> 6400 <SEP> 0
<tb> 778 <SEP> 6400 <SEP> 0
<tb> 923 <SEP> 6400 <SEP> 0
<tb> 926 <SEP> 6400 <SEP> 0 The viremia is measured by quantitative hybridization and represents the total amount of virions released during the follow-up period.

All ducklings born from canines immunized with pCI-preS / S and having a anti-preS antibody titre> 6400 are protected against a high-titre DHBV viral challenge (Table 1, 2nd line).

On the other hand, all ducklings born from canines immunized with pCI were infected (Table 1, the first line).

All of these results show that the method for producing antibodies in the yolk by immunization with naked DNA coding for an appropriate antigen, according to the invention, is easy to implement and allows to produce in large quantities, in the new ones, antibodies specific for this antigen and its variants and which have a high neutralizing power and are highly protective.

Claims (11)

1. A method of producing antibodies in egg yolk characterized in that it comprises - the immunization of avians by a naked DNA, - the collection of eggs then egg yolks, and - optionally the purification of the eggs. antibodies produced in the yolks. 2. Method according to claim 1, characterized in that the immunization is carried out in the hen or cane. 3. Method according to claim 1 or 2, characterized in that the immunization is carried out by a plasmid encoding an antigen or a protein selected from antigens and proteins of prokaryotic and eukaryotic origin. 4. Use of at least one antibody obtained by a method according to any one of claims 1 to 3, said antibody being optionally combined with a suitable substance, for the preparation of an immunological reagent that can be used to screening, diagnosis and / or monitoring of diseases in humans or animals. 5. A method for the detection, diagnosis and / or monitoring of a disease, in humans or in animals, of detecting the presence of an antigen in a biological sample, characterized in that it involves said sample is present with at least one suitable antibody produced by a method according to any one of claims 1 to 3. 6. Kit or diagnostic kit for the detection, diagnosis and / or follow-up of a disease, in humans or animals, characterized in that it uses at least one antibody obtained by a method according to any one of claims 1 to 3, suitable reagents and means for detecting antigen-antibody binding formation. 7. Use of egg yolks, or egg yolk fractions containing the antibodies produced by a method according to any one of claims 1 to 3 or purified antibodies produced by a method according to any one of claims 1 to to 3, for the preparation of a medicament useful in human or veterinary medicine. 8. Use according to claim 7, characterized in that the drug is useful in immunotherapy. 9. Use according to claim 8, characterized in that the drug is intended for passive immunization against enteric pathogens or oral infections, in humans or in animals. 10. Use according to claim 9, characterized in that the drug is capable of treating an infection selected from the group consisting of viral infections, bacterial and protozoal infections. 11. Use according to claim 8, characterized in that the drug is capable of treating diseases selected from the group comprising cancers and allergies.