WO2008004902A2

WO2008004902A2 - Protein, their fragment and use thereof

Info

Publication number: WO2008004902A2
Application number: PCT/PL2007/000045
Authority: WO
Inventors: Andrzej Gamian; Danuta Witkowska; Bernadeta Szostko
Original assignee: Instytut Immunologii i Terapii Doswiadczalnej PAN; Instytut Przemyslu Farmaceutycznego
Current assignee: Instytut Immunologii i Terapii Doswiadczalnej PAN; Instytut Przemyslu Farmaceutycznego
Priority date: 2006-07-04
Filing date: 2007-07-04
Publication date: 2008-01-10
Anticipated expiration: 2009-01-04
Also published as: PL380105A1; WO2008004902A3

Abstract

The isolated protein present in the extract of bacterial outer membrane proteins and its fragments suitable for using in medicine and pharmacy, especially for producing vaccines and diagnostic tests.

Description

Protein, their fragment and use thereof

The subject of this invention is method of isolation of diagnostic preparation, which is bacterial protein purified from bacterial mass of enterobacteria and also the method of its using in test for the determination of specific anti enterobacterial immunoglobulin deficiencies as an indicator of antibacterial immunity, which play a main role in pathogenesis of infectious diseases, by direct using of this protein preparation for the determination of specific anti-enterobacterial antibodies in human sera, also the method of using of this preparation in affinity chromatography for isolation of specific protective immunoglobulins as a therapeutic agent in immunodeficiencies, and also the method of using this preparation as a protein carrier in conjugate vaccines. Outer membrane proteins (OMP) of Gram-negative bacterial cell wall participate in maintaining of bacterial cell integrity, its adaptation to the environment and in interactions with host cells [Koebnik R. et al. MoI. Microbiol, 2000, 37, 239-253]. These proteins named porins, exposed on the bacterial cell surface, are immunologically important molecules in many Gram-negative bacteria. These proteins may affect the physiological functions of tissues and participate in mechanisms of pathogenicity, progression of infections and in development of inflammatory response [Lin J. et al. Microbes and Infection, 2002, 4, 325-331]. In many inflammation processes caused by Gram-negative as well as Gram-positive bacteria, antibodies against bacterial proteins were found [Biswas T., FEMS Immunol. Med. Microbiol. 2000, 29, 129-136]. These specific features of bacterial proteins (OMP) and their accessibility to the host immune system make them attractive as components of vaccines, convenient carriers for carbohydrate antigens and also as immunodiagnostic markers [Roy S. et al., Infect. Immun. 1994, 62, 4333-4338]. Shigellosis, salmonellosis and other diseases caused by representatives of Enterobacteriaceae family are still very important medical problem, especially in developing countries. In our earlier studies on OMP from different serotypes of S. flexneri [Witkowska

D. et al. FEMS Microbiology Letters 1982, 13, 109-111], S. sonnei [Adamus G. et al. Arch. Immunol. Ther. Exp. 1986, 34, 513-516] and Hafnia alvei [Witkowska D. et al. Arch. Immunol. Ther. Exp. 1988, 36, 711-715] we have shown almost identical electrophoretic profile of OMPs with two characteristic 33 K and 36 K major proteins [Witkowska D. et al. FEMS Microbiology Letters 1982, 13, 109-111]. hi the outer membrane of S. sonnei a heat modifiable 33 K protein and two other, 35 K and 37 K associated with peptidoglycan proteins were present [Adamus G. et al. Arch. Immunol. Ther. Exp. 1986, 34, 513-516]. Moreover, we also observed the distinct immunogenic and protective properties of OMPs extracts from Salmonella, Shigella and Hafnia [Adamus G. et al. Infect. Immun. 1980, 30, 321-324; Mulczyk M. et al. Arch. Immunol. Ther. Exp. 1981, 29, 85-90; Mulczyk M. et al. Arch. Immunol. Ther. Exp. 1984, 32, 631-63512]. Immunization of animals with OMP from different serotypes of Shigella induced the cell and humoral mediated immunity, protected guinea pigs against keratoconjunctivitis shigellosa induced by homological and heterological strains of bacteria, protected mice for a long time against lethal dose of homological and heterological strains of bacteria [Adamus G. et al. Infect. Immun. 1980, 30, 321-324; Witkowska D. et al. Arch. Immunol. Ther. Exp. 1985, 33, 625-628; Witkowska D. et al. Arch. Immunol. Ther. Exp. 1986, 34, 499-504]. Complete protection against bacterial challenge was observed on fourth day after immunization, and the highest level of specific antibodies against OMP was observed on 10th-21th days after immunization. It was also shown that immunity in mice induced with OMP may be transferred passively to nonimmunized animals by the serum of immunized animals [Witkowska D. et al. Arch. Immunol. Ther. Exp. 1986, 34, 499-504]. It was also found that OMPs isolated from Shigella flexneri exhibit immunomodulatory properties, as small doses of OMPs stimulated delayed hypersensitivity to sheep red blood cells, whereas higher doses suppressed this type of immune response. Immunochemical analysis of OMPs from Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei performed by Roy et all. showed similar electrophoretical profiles in all Shigella species with the major protein bands around 34-38 kDa, indicating them to be porins and confirmed our earlier results [Roy S et al. Microbios, 1994, 79, 318, 55-64]. Analysis of antigenic properties of OMPs from four Shigella species showed cross-reactivity of their major protein with molecular mass 38 kDa. In sera of mice immunized with formalinized bacteria of four Shigella species, specific antibodies of IgM and IgG class were shown, but in IgG class dominating was IgGl subclass [Roy S et al. Microbios, 1994, 79, 318, 55-64]. Immunoglobulins are important effectors of specific humoral immunity. Different classes and subclasses of immunoglobulins exhibit distinct functions. The predominant subclasses of immunoglobulins G (IgG) elicited during natural infection could influence the capacity of the humoral response to provide an adequate defense of the host. Bacterial proteins are the antigens preferentially inducing IgGl response, with minor contribution of IgG3 and IgG4 [Islam D. et al. Infect. Immun. 1995, 63, 2054-2061]. It was reported that acquired immunity to Shigella bacilli is mainly connected with the production of local secretory immunoglobulin A (IgA) and serum IgG, which are specific to some bacterial virulence proteins, which are the major bacterial cell surface components [Sansonetti P. and Phalipon A., Res. Immunol. 1996, 147, 595-602]. hi our earlier studies we observed immunogenic and protective properties of OMP from Shigella, Salmonella and Hafiiia [Mulczyk M. et al. Arch. Immunol. Ther. Exp. 1981, 29, 85-90]. In an animal model of shigellosis it was shown that the 38 kDa protein and three other outer membrane proteins were the major antigens in the induction of protective immune response [Witkowska D. et al. Arch. Immunol. Ther. Exp. 1992, 40, 119-124] and thus these proteins may be the excellent candidates as antigens or carriers in conjugate vaccines and may serve as immunodiagnostic markers. In order to investigate the reactivity of OMP with human sera from healthy donors and of immunodeficiency patients, we isolated these OMP from cell wall of Shigella flexneri bacilli and few other Gram-negative bacterial strains. Interestingly, we observed in immunoblotting the reactivity of OMPs with umbilical cord plasma. This important observation of such beneficial reactivity indicates that cord plasma antibodies may be protective, because antibacterial protective antibodies are transferred from the mother to fetus. That reactivity was present in all examined children, adolescents and adults sera. Specific antibodies of IgA and IgG classes interacted primarily with 38 kDa protein, in similar way for several studied enterobacterial strains, but different for Pseudomonas aeruginosa from Pseudomonadaceae family. This indicates for the specificity towards Enterobacteriaceae strains, the major components of intestinal microbial flora, which translocate to the circulation and are responsible for the development of antibacterial immunity. Reactivity was then determined of sera of several groups of children with 38 kDa protein from Sh. flexneri purified by preparative electrophoresis. The reactivity of this protein with sera in ELISA was age dependent, decreased in infants below 4 months of life, increased from 12 months of life to 5 years and next in teenagers the ratio of reactivity became like in adults. These beneficial results show that 38 kDa protein is a major enterobacterial antigen recognized by human immune system and plays a protective role against enterobacterial infection. This allows to expect that this protein is non-toxic, non-pathogenic and may be useful as an antigen carrier in construction of conjugate vaccines. The specific reactivity of IgG and IgA estimated as normal, physiological is transferred from the mother to blood stream of fetus and thus protects newborn to infection during the first period of his life. The subject of this invention is bacterial surface protein beneficially recognized by antibodies of healthy human without active infection, what indicates that correct level of protective antibodies is very important. The OMP preparations were obtained from Shigella flexneri strain and other Gram-negative strains, and reactivity of human sera was determined with 38 kDa protein isolated from Shigella flexneri strain 3a. Oczyszczone korzystnie z preparatu bialkowego otrzymanego z masy bakteryjnej bialko, bξdajce przedmiotem wynalazku, bylo obecne w 27 szczepach nalezajcych do 9-ciu gatunkόw bakterii reprezentuja_cych rodzinς Enterobacteriaceae. Wydajnosc preparacji mogq. charakteryzowac przykladowo wyniki, gdzie z 90 g masy bakteryjnej Shigella flexneri 2a uzyskano 535 mg preparatu bialek blonowych (0.60%), z 85 g Shigella flexneri 3a otrzymano 465 mg bialek (0.55%) a ze 120 g masy bakteryjnej Hqfhia alvei uzyskano 900 mg bialek (0.75%). Wydajnosc preparacji (OMP) z masy bakteryjnej 9 serotypόw Sh. flexneri wahala siζ w granicach od 0.3 % do 0.8%. Otrzymane preparaty bialek charakteryzowano w elektroforezie na zelu poliakrylamidowym w obecnosci SDS i stwierdzono, ze w przypadku Shigella flexneri sa_ to mieszaniny bialek, zrόznicowane w zaleznosci od serotypu. Jednakze, sposrόd dwόch glόwnych bialek o cifzarze czajsteczkowym 35 kDa i 38 kDa, charakterystycznych dla wszystkich badanych serotypόw rόznych gatunkόw z rodziny Enterobacteriaceae takich jak Hafnia alvei i Salmonella, lecz nie u Pseudomonas aeruginosa, stwierdzono wyraznq. immunoreaktywnosc przeciwcial typu IgG i IgA w surowicach ludzi zdrowych tylko z jednym z glόwnych bialek (OMP), o ciςzarze czajsteczkowym 38 kDa. Immunoreaktywnosc taka wystξpowala w przypadku bialek ze szczepόw: Sh. flexneri, Klebsiella pneumoniae, Escherichia coli, Citrobacter, Proteus i Hafnia, z mniejsza_^ intensywnoscia_^ w przypadku bialek Sh. sonnei. Enterobakteryjne bialko 38 kDa bylo rozpoznawane przez przeciwciala klasy IgG, IgM i IgA obecne w surowicach ludzkich i co jest bardzo wazna_^ nowoscia_, stwierdzano wyraznq. reaktywnosc przeciwcial klasy IgG osocza krwi pζpowinowej z tym glόwnym bialkiem. Dotychczas badacze analizowali bialka OMP w aspekcie wlasciwosci ochronnych na modelu zwierzφcym w zakazeniach i rzadziej na surowicy ludzkiej w fazie chorobowej i w czasie rekonwalescencji, natomiast nasze podejscie polegalo na analizie krwi pζpowinowej, surowicy dzieci i ludzi zdrowych. Ta ostatnia reaktywnosc w krwi pζpowinowej wskazuje na ochronna_ rolς swoistych przeciwcial skierowanych na immunogenny epitop bakteryjny, ktόry nie powinien bye szkodliwy dla czlowieka. Otrzymane rezultaty sklonily nas do oczyszczenia tego bialka i przeprowadzenia jakosciowych badaή jego immunoreaktywnosci z innymi surowicami ludzi doroslych i dzieci w rόznym wieku, takze pacjentόw z niedoborami immunologicznymi. Badania strukturalne tego bialka metoda_^ spektrometrii masowej fragmentόw trypsynowych i ich identyfikacja_^ z odpowiednimi sekwencji w bazach danych wykazaly jego tozsamosc z bialkiem OmpF E. coli. Istotq. wynalazku jest izolacja immunoreaktywnego bialka 38 kDa na przyklad z Sh. flexneri, korzystnie stosujajc elektroforezς preparatywnq. na zelu poliakrylamidowym w obecnosci SDS i elucjς cia^la^ oraz opracowanie korzystnie testu immunoenzymatycznego. Do testu uzywa siζ wyizolowanego bialka korzystnie oplaszczonego na plytkach. Test okazal siς przydatny w celu potwierdzenia roli bialka OMP-38 we wrodzonej i nabytej odpornosci, gdyz poziom reaktywnosci przeciwcial klasy IgG i IgA jest znaczajco nizszy u dzieci niz u doroslych, zgodnie z klasycznym obrazem rozwoju odpornosci. W przypadku surowic pochodzajcych od pacjentόw z niedoborami IgA i IgG, w obydwu grupach poziom swoistych przeciwcial byl znaczajco nizszy. It should be emphasized that crude preparations of outer membrane proteins isolated from cells of Sh. flexneri, K. pneumoniae, Hafnia and Citrobacter comprise a multi-component mixture of proteins. The efficient method for purification of 38 kDa protein from Sh. flexneri appeared the preparative electrophoresis, beneficially one step process. Similarity between bacterial antigens and host antigens, called molecular mimicry, is considered as one of pathogenic factors in many autoimmunological diseases.

Knowledge on the occurrence of such similarity between bacterial antigens and their host antigens, allows to exclude some bacterial proteins as potential components of safe and

• effective antibacterial vaccines. Identification of anti 38 kDa protein antibodies in cord blood plasma indicates that the mimicry phenomenon does not occur here and the protein may be used as an early marker of innate immunity, which is especially important for protection against intestinal bacilli. Moreover, bacterial protein 38 kDa may be considered as a potential marker for detection of immune deficiencies, and also as indicator used for monitoring of development of antibacterial immunity, also during therapy. Our observations indicate for the protective role of antibodies anti 38 kDa OMP. Such antibacterial protection is transferred from mother to fetus. The level alters of protective antibodies anti 38 kDa OMP and depends on age, what corresponds to classic pattern of development of immune response, resembling the development of antibacterial immunity typical to humoral response. The presented invention permits beneficially to prepare the affinity column with chemically linked OMP 38 kDa protein and using this column enables beneficially purifying the specific antibodies from animal and human origin of anti enterobacterial specificity. Such antibodies have diagnostic and therapeutic value. Preparations of human immunoglobulins may be applied to the affinity column, thus producing beneficially the fraction of specific anti enterobacterial antibodies which bind to the affinity gel and also beneficially the fraction of non bound immunoglobulins with total value activity, for example antiviral, for further therapeutic applications. The column is used beneficially for several times. The use of substitutions with high doses of immunoglobulins may be harmful [Bernatowska E., Post. Hig. Med.Dosw., 2002, 56, Supl., 23-31], and the present invention permits beneficially for significant lowering of effective doses of immunoglobulin substitutions. There is urgent need for administering the preparations of specific antibacterial immunoglobulins to newborns [Gajewska E. et al, Post. Hig. Med. Dosw. 2002, 56, Supl., 103-126], children and adults in several cases [Wrόbel G. et al., Post. Hig. Med. Dosw. 2002, 56, Supl. 59-68 and other articles in his volume], therefore the prezent invention will have broad application in therapy.

The present invention, thanks to obtained results of studies, enables beneficially also to apply the 38 kDa OMP protein as carrier for conjugate vaccines, useful and safe for its use as active for inducing the formation of protective antibodies. E x a m p l e s

1. Preparation of extract of bacterial outer membrane proteins (OMP).

Wet bacterial mass from 7 hours culture in liquid BHI medium at 37°C was obtained by centrifugation and washing with 1OmM Tris-HCl buffer pH 7.6, containing 10 mM MgSO₄. Bacteria were suspended in the same buffer, containing 20 μg of RNase and 20 μg of DNase per ml, and then disrupted in an ultrasonic disintegrator for 10 min. The disrupted cell suspension was centrifuged at 7000 x g to remove undisrupted cells, and then the resulting supernatant was centrifuged at 150,000 x g for 1 hour to separate the envelope fraction. The cell envelope sediment was extracted twice with 10 mM Tris-HCl, pH 7.6, containing 10 mM MgSO4 and 2% Triton X-100 at room temperature to dissolve the cytoplasmic membrane. After centrifugation at 150,000 x g, the sediment was extracted twice with the same buffer containing 2% Triton X-100 and 5 mM EDTA. The supernatant obtained after centrifugation at 160,000 x g contained OMPs, which were precipitated with 2 volumes of 95% ethanol. The OMP fraction was analysed by SDS-PAGE which showed to contain about 20 proteins. Fractions OMP contained less than 5% lipopolysaccharide (LPS), as calculated from 3-deoxyoctulosonic acid measurement.

2. Preparation of 38 kDa protein immunoreactive with human serum.

The preparative electrophoresis of the outer membrane proteins was performed beneficially using Prep Cell 491 apparatus (BioRad) with a 37-mm ID tube and 80 ml of 10% or 12.5% resolving gel and 20 ml of 5% stacking gel and buffer containing 25 mM Tris, 0.192 M glycine, and 1% SDS, pH 8.3, for both electrophoresis and elution. After application of the 30- to 40-mg sample of OMPs extract to the top of the stacking gel, electrophoresis was run at the maximum setting of 260 V and 109 mA. Elution was started when the bromophenol blue indicator band reached the base of the separating gel. Fractions of 1.4 ml were collected according to the protein, continuously monitored with an UV detector set at 280 nm, and checked with SDS-PAGE and immunoblotting. Fractions beneficially with the appropriate proteins were dialyzed against water, pooled, and concentrated using vacuum centrifugation. The OMP fractions were characterized in polyacrylamide gel at reducing conditions using 10% or 12,5% gels with standard methods.

3. Determination of the level of specific anti enterobacterial immunoglobulins in human serum.

The level of protective antibodies against enterobacteria was determined in human serum with ELISA test on plates coated with protein antigen. The polystyrene 96-wells plates were coated with a solution of the 38 kDa OMP from Shigella flexneri 3a cell wall (1 μg/100 μl antigen) in carbonate buffer, pH 9.6, at 37⁰C for 3 h and then at 4⁰C overnight. The plates were then blocked with 1% BSA in water. After blocking, wells were washed three times using 250 μl TBS-T per well (TBS-T is a solution of 20 mM Tris-HCl, 50 mM NaCl buffer, pH 7, containing 0.05% Tween 20). The wells were then filled with serial dilutions of human sera and the plates were left at room temperature for 2.5 h. After washing three times with TBS-T, 100 μl of an alkaline phosphatase-labeled goat anti- human IgG or IgA diluted 1:10,000 in PBS was added to each well. Following incubation at room temperature for Ih, the plates were washed three times with TBS-T (250 μl/well) and then 200 μl/well of pNPP substrate (Sigma) was added. After 30 min the reaction was stopped by 50 μl of 3M NaOH and the optical density was read at 405 run using a Dynatech MR 5000 Microplate Reader. 4. Fractionation of antibodies with the method of affinity chromatography.

The affinity chromatography column was prepared, namely with bound 38 kDa protein. Agarose gel, for example Sepharose 4B (Pharmacia) (10-20 ml) was activated with CNBr (Fluka) according to the standard procedure, preferentially for about 30 min at room temperature at a constant pH of 11. The excess of cyanogen bromide was washed with water and 0.1 M NaHCO₃ of pH 8.2. To the activated gel the protein 38 kDa OMP was added (30 mg) in 5 ml 0.1 M NaHCO₃ of pH 8.2 and gel was incubated for 2 hours with gentle rotation. Then the mixture was left at room temperature for 2 hours with equal volume of 1 M ethanolamine (POCh)₅ and then at 4 ⁰C overnight with gentle rotation. The gel was washed with water and left for 30 min with 2 M K₂HPO₄, then with water again. Finally, glass column (1 x 10 cm) was filled with gel suspended in the PBS without Mg²⁺, Ca as eluent. Serum (20 ml) diluted two fold with PBS was precipitated with small portions of ammonium sulfate (9.6 g) and incubated at 4°C for 2 hours. Then the pellet was centrifuged at 3000 r/min for 30 min at 4°C. The pellet was solubilized in 10 ml PBS and dialyzed to PBS at 4⁰C with several changes of buffer. The salted-out antibodies were concentrated by ultrafiltration to a volume of 3 ml Then the salted-out antibodies were fractionated of the affinity column with immobilized protein. Antibodies were applied onto the gel and column was washed with PBS without Mg²⁺, Ca²⁺. Fractions 2 ml were collected and checked for the protein content by measuring the absorbance at 280 nm. After washing out the proteins not bound with gel, the antibodies of low affinity to the antigen were eluted with 1 M NaCl in PBS without Mg²⁺, Ca²⁺. The fraction of antibodies of high affinity to immobilized antigen to the gel was obtained by elution with using 3M KCNS (Reachim) in PBS without Mg²⁺, Ca²⁺. Finally, column was thoroughly washed with PBS without Mg²⁺, Ca²⁺. Material bound to the gel and then eluted was dialyzed to several changes of PBS and concentrated. Specific antibodies were concentrated to a volume of about 1 ml and stored at -2O⁰C in 50% glycerol. 5. Preparation of conjugate

Antigen or hapten, for example carbohydrate (2-4 mg) and bacterial protein OMP 38 kDa as protein carrier (1-2 mg) were mixed together in water (about 0.2ml), lyophilized, and then heated at a temperature up to 130°C for several tens minutes, beneficially for 15-

60 minutes. After the reaction the product was solubilized and dialyzed to PBS, and stored at -20°C.

Claims

Patent claims

1. An isolated protein occurring in extract of bacterial outer membrane proteins possessing molecular mass about 38 kDa, as well comprising the amino acid sequence which contains not less than 80% of identity to any sequence shown on fig. 1.

2. The protein of claim 1 wherein it is isolated from bacteria belonging to the family of Enterobacteriaceae.

3. The protein of claim 1 wherein it was isolated from bacteria belonging to the species Shigella flexneri.

4. A fragment of protein of claims 1-3 wherein it possesses an amino acid sequence selected among sequences underlined on fig. 1 but longer than 4 amino acids.

5. The application of protein of any claims 1 to 3 or its fragment of claim 4 for the producing of immunochemical tests.

6. The application of claim 5 wherein the produced tests serve for the analysis of specific antibodies in immunological deficiencies.

7. The application of protein of any claim of 1 to 3 or its fragment of claim 4 for producing of vaccines.

8. The application of claim 7 wherein the protein or its fragment are utilized for the producing of conjugate vaccines, beneficially as carriers.

9. The application of protein of any claim from 1 to 3 or its fragment of claim 4 for producing of carriers for affinity chromatography.

10. A method for producing of enterobacterial outer membrane proteins with molecular mass abort 38 kDa wherein from the extract of outer membrane proteins obtained from bacterial mass of Enterobacteriaceae, beneficially Shigella flexnerii, is obtained a single protein by preparative electrophoresis in denaturing conditions.

11. Affinity columns wherein they have the protein of any claim from 1 to 3 or its fragment of claim 4 covalently bound to chromatographic gel.

12. The metod of producing of animal antibodies protective against enterobacteria wherein the animals are immunized with protein of any claim from 1 to 3 or its fragment of claim 4 and from the obtained serum an immunoglobulin fraction is salted out, beneficially with ammonium sulfate.

13. The metod of producing therapeutic specific antibodies protective against enterobacteria wherein from human serum or from preparation of human immunoglobulins or from fraction of animal antibodies obtained with the metod of claim 12 are isolated on the affinity column of claim 11 a specific antibodies.

14. The metod of producing diagnostic conjugates or conjugate vaccines wherein protein of any claim from 1 to 3 or its fragment of claim 4 is covalently linked as carrier protein with antygen or hapten.