CA2074585A1 - Monoclonal antibodies directed against streptococcus pneumoniae - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Streptococcus pneumoniae is a major cause of systematic bacterial infections such as pneumonia, meningitis, otitis and bacteremia. Such infections can be rapidly diagnosed using a monoclonal antibody which is specific against the bacterium, and in particular against surface accessible proteins having molecular masses of 40-kDa and 67-kDa.

Description

2~ 35 BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
-This invention relates to a monoclonal antibody (MAb) directed against non-capsular somatic antigens of Streptoccoccus pneumoniae ~S. pneumoniae).
S._pneumoniae is a major cause of systemic bacterial infections in humans, especially infants, the elderly or immunocompromised persons. It is the bacterium most frequently isolated from patients with bacterial pneumonia.
S. pneumoniae is also frequently isolated from patients with meningitis, otitis and bacteremia. The rapid diagnosis of pneumococcal inections, in particular meningitis which has a high fatality rate, is of critical importance. Reliable diagnostic methods and reagents for defining the causes of infections are needed for planning both treatment and prevention strategies.
DISCUSSION OF THE PRIOR ART
Despite its prevalence, diagnosis of pneumococcal disease is still a problem. The gold standard, blood culture is unique in specificity, but has a low degree of sensitivity~
and is of little value when the samples are taken for culture after antibiotic treatment has already started. A major difficulty in devising a diagnostic assay for pneumococcal disease is the multiplicity of serotypes S. pneumoniae. The -25 bacterium can be divided into at least 85 serotypes, and, for the most part, pneumococcal pneumonia, otitis media and ~ 2~ 5 meningitis are caused by approximately 20 serotypes. Most of the diagnostic tests previously developed used pooled anti-capsular serum for the detection of pneumococcal antigens.
Such a pooled reagent suffers from two disadvantages, namely S (i) it is not sufficiently sensitive and (ii) several cross-reactions, particularly with other streptococci, are associated with the antiserum. 5imilarly, antisera raised to the pneumococcal C-polysaccharide, a common antigen, have also been shown to cross-react with non-pneumococcal strains of bacteria.
Therefore, there remains a need for a test which can be used for the early diagnosis of pneumococcal infections, and which is specific while possessing a high degree of sensitivity.
The present invention is based on an appreciation by the inventors that an alternative approach to the detection of capsular antigens would be the identification of epitope common exclusively to all pneumococci. There is an ongoing search for specific immunodiagnostic markers or tests to aid in the early diagnosis of pneumococcal and other infections.
The development of monoclonal antibody technology by Kohler and Milstern [Nature 256, 52(1975)] provided an enormous opportunity for advancement in the diagnosis of infectious diseases. Monoclonal antibodies can be used for diagnostic purposes, because they demonstrate the presence of an antigen specific for a given species of microorganism. However, until , '` ;2Q79 ~:
the research described herein was conducted by the present inventors, the need for a specific and sensitive test for pneumococcal infections still existed.
GENERAL DESCRIPTION OF THE INVENTION
. . . _ An object of the present invention is to meet the above need by providing a monoclonal antibody (MAb) directed against non-capsular somatic antigens produced exclusively by S. pneumoniae isolates.
Another object of the invention is the provision of a continuous hybridoma cell line which elaborates and secretes specific and homogeneous MAbs to species - specific proteins f ~ _E~ . Such MAbs are reactive with epitopes on pneumococcal proteins, especially proteins of about 40-kDa and about 67-kDa.
Accordingly, the present invention relates to a monoclonal antibody which specifically reacts with surface accessible protein antigens of the bacterium Streptococcus pneumoniae.
One or both of the above described MAbs can be used in a diagnostic method for the detection of the 40-kDa and/or the 67-kDa proteins. The MAbs may detect pneumococcal antigens in samples using known immunology techniques, including enzyme-linked immunosorbent assay (ELISA), biligand binding (sandwich technique), radioimmunoassay (RIA), agglutination, immunocytopathology and flow cytometry. The MAbs may be immobilized on an inert surface, embedded in a .' ' ~

gel, or many be con~ugated to a molecule that imparts color, fluorescence or radioactivity to the MAbs. The MAbs may also be used to isolate and purify the 40-kDa or 67-kDa protein antigens.
DESCRIPTION OF PREFERRED EMBODIMENT
Production of_monoclonal Antibodies _MAbs) irected Aqainst Pneumococcal Proteins Bacterial Strains A total of 128 strains of S. pneumoniae representing 30 capsular serotypes were obtained from the following:
Laboratoire de la Sante Publique du Quebec, Sainte-Anne de Bellevue, Canada; Children's Hospital of Eastern Ontario, Ottawa, Canada; Sainte-Justine Hospital, Montreal, Canada and Caribbean Epidemiology Centre, Port of Spain, Trinidad. The lS 83 non-pneumococcal bacterial isolates (listed in Table l) representing 22 genera and 44 species from the stock collection of the National Laboratory for Immunology were included in the study.

.5 Non-pneumococcal Isolates Tested Genus Species Number of Strains Tested Streptococcus pyogenes 2 agalactiae 3 dysagalactiae 2 bovis 2 mitis 2 acidominimus mutans salivarius 3 sanguis 10 suis 9 Enterococcus faecalis faecum Gemella morbillorum hemolysans Staphylococcus epidermidis 2 : aureus Bacillus Escherichia coli 2 Klebsiella pneumoniae 2 Neisseria meningitidis 8 lactamica 2 mucosa cinerea perflava 2 flava flavescens gonorrhoae subflava Haemophilus influen~ae 3 Moraxella catharrhalis Alcaligenes odorans Flavobacterium odoratum Citrobacter freudii '``' `~

,~

~7 ~35 TABLE 1 ~con'd) Non-pneumococcal Isolates Tested Genus Species Number of Strains Tested Pseudomonas aeruginosa Enterobacter cloaca aerogenes Proteus vulgaris Providencia rettgeri Serratia marcescens Salmonella thyphimurium Shigella flexneri sonnei Edwarsiella tarda Xanthomonas maltophilia Antiqen Preparations .

Various S. pneumoniae antigens were prepared for immunization and immunoassay. Heat-killed whole cell antigens were obtained by incubating bacterial suspensions in a water bath prewarmed at 50~C for 20 min. In order to ; prepare formalin-killed whole cell antigen, bacteria were suspended in 1% formalin ~final concentration) for 18 h at 4~C. Detergent-soluble proteins were extracted from S.
Eneumoniae strain Tri (Trinidad) 810062 as follows. Heat-; killed bacteria were suspended in 10 mM Hepes buffer (4-(2-Hydroxyethyl)-l-piperazinethan-sulfonsaure; Boehringer Mannheim GmbH, Germany), pH 7.4, and sonicated at 20,000 Kz/sec, four times for 30 sec (Vibra cell Vc600; Sonics &

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5!35 Materials Inc., Donbury, Connecticut). Intact cells and large debris were removed by centrifugation at 1,700g for 20 min.
The supernatant was collected and centrifuged at 100,000g for 60 min. ~he pellet was resuspended in 1 ml of Hepes buffer, and 1 ml of N-lauroyl sarcosine (Sigma Chemical Co., St-Louis, Mo.) was added. The mixture was incubated for 30 min at room temperature and the detergent-soluble fraction was harvested by centrifugation at 100,000g for 60 min. To prepare sonicated antigen, S. pneumoniae strains were suspended in phosphate-buffered saline (PBS) and disrupted by sonication for 5 min three times. Sonicates were centrifuged at 48,000g for 20 min and ~he supernatant was collected and filtered through 0.22 m filters (Gelman Sciences, Ann Arbor, Mi.).
The protein contents of antigen preparations were determined by the BIO-RAD protein assay (Bio-Rad Laboratories, Missaussauga, Ontario, Canada). BIO RAD is a registered trademark.
Whole cell extracts were prepared for SDS-PAGE and Western blot analysis by adding 10~(w/v) SDS to cell suspensions containing 108 organisms per ml. The bacterial suspensions were then sonicated on ice for 2 min and boiled for 5 min.
Pr~duction of Monoclonal Antibodies Female BALB/c mice (Charles River Laboratories, St-Constant, Quebec, Canada) were immunized with S. pneumoniae antigens. One set of mice (fusion experiment 1) were 2~ s immunized by intraperitoneal injections with 107 formalin-killed whole cell antigen from strain SJH-l suspended in Freund complete adjuvant tGibco Laboratories, Grand Island, N.Y.) and were boosted at two-week intervals with the same antigen and then with sonicated antigen (S ~g protein) from homologous strain SJH-l in Freund incomplete adjuvant.
Fourteen and three days before fusion, mice were boosted with sonicated antigen suspended in PBS. A second group of mice (fusion experiment 2) were immunized three times at three-week in~ervals with 5 ~g of detergent-soluble pneumococcal proteins extracted from strain Tri 810062. The antigen was mixed with 25 ~g of Quil A (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) prior to injection. Five and three days before fusion, mice were injected intraperitoneally with lS antigen suspended in PBS.
Hybridomas were produced by fusion of spleen cells recovered from immunized animals with nonsecreting SP2/0 or P3-X63-Ag 8.653 myeloma cells as previously described [Hamel et al., J. Med. Microbiol. 25, 2434 (1987)]. Specific hybridoma were cloned by sequential limiting dilutions, expanded and frozen in liquid nitrogen. The class, subclass, and light-chain of MAbs were determined by ELISA as reported elsewhere [Martin et al, Eur. J. Immunol 18, 601 (1988)] using reagents obtained from Southern Biotechnology Associates Inc., Birmingham, Alabama.

Enz~me Immunoassay Procedures The ELISA procedure was performed as described by Brodeur et al ~Infect. Immun. 50, 510 (1985)]. Coating antigens were heat-killed whole cell antigens (approximately 107 organisms per ml) in 0.05 M carbonate buffer, p~ 9.6.
Alternatively, sonicated antigens or detergent-soluble proteins were used at a concentration of 7.5 ~g per ml.
Dot-blot immunoassay was used to screen the MAbs against a large number of bacterial strains [Martin et al, Infect Immun. 59, 1457 (1991)]. Heat-killed whole cell antigens were blotted onto nitrocellulose, blocked with PBS
containing 3% (w/v) bovine serum albumin (BSA), and then incubated sequentially with MAbs and peroxydase-labelled secondary antibody.
SDS-PAGE and Immunoblot Analysis Whole cell extracts were resolved by SDS-PAGE
according to the method of Laemmli [Nature 227, 680 (1970)~
with some modifications [Martin et al, Eur. J. Immunol. 8, 601 (1988)]. After separation, the proteins were stained with Coomassie blue. Following SDS-PAGE, Western blot analysis was performed by the method of Towbin et al [Proc. Natl. Acad.
Sci. US~ 76, 4350 (1979)].
~ Enzymatic and Periodate Treatments of Pneumococcal Anti~ens S. pneumoniae antigens were treated with various proteolytic enzymes and sodium periodate in order to determine 2~ 5 which components, proteins or carbohydrates, were reactive with MAbs. Detergent-soluble proteins extracted from S. pneumoniae strain Tri 810062 were transferred electrophoretically from polyacrylamide gels to nitrocellulose papers and then treated for 2 h at room temperatuxe with either proteinase K (15 units per ml; Sigma Chemical Co., St.
Louis, Mo.), trypsin (180 units per ml; Sigma), or chymotrypsin (25 units per ml; Sigma). Periodate oxidation was performed as described before ~Martin et al, Infect.
Immun. 28, 1720 (1990]. Blots were treated with 100 mM sodium periodate for 1 h at room temperature in the dark. The immune reactivity of MAbs with untreated and treated blots were thereafter tested by enzyme immunoassay as described above.
Antibody Accessibility Radiommunoassay Adsorption of MAbs at the surface of live bacteria was performed [Martin et al, (1988); Proulx et al, ~1991)] in order to determine whether MAbs were directed against surface exposed epitopes. Briefly, hybridoma culture supernatant was mixed with 109 live intact bacteria strains and incubated for 2 h at 4C. After three washes with PBS, the bacterial cells were suspended in a solution containing 125iodine-labelled anti-mouse immunoglobulins (ICN Biomedicals Inc., Costa Mesa, California) and incubated for 1 h at room temperature. The bacteria were washed twice with PBS and the amount of cell-bound 125ioaine radioactivity was determined.

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Properties of MAbs Culture supernatants of hybridomas obtained fromfusion of primed spleen cells and myeloma cells were screened initially by ELISA using sonicated antigens and heat-killed whole cell antigens (fusion experiment l) or detergent-soluble proteins (fusion experiment 2) prepared from the homologous strain. In order to recover the hybridomas secreting S. pneumoniae-specific MAbs, each positive hybridomas was further tested by dot-enzyme immunoassay and Western blotting against several antigenic preparations obtained from S. pneumoniae strains from various serotypes and from non-pneumococcus strains. In this manner, two hybridomas secreting antibodies that reacted with S. pneumoniae strains were selected and subcloned twice by limiting dilution. ELISA
analysis revealed that both hybridomas, Sp-5 (fusion experiment l) and Sp-8 (fusion experiment 2), secreted IgGlkappa immunoglobulins. Western immunoblotting demonstrated that MAb Sp-5 and MAb Sp-8 recogniæed a protein band with an apparent molecular mass of 40-kDa and 67-kDa respectively.
MAb_Reactivity with Sodium Periodate and Proteolytic Enzyme Tr_ated Antigens Sodium periodate and enzyme treatments were used to confirm the protein character of the epitopes. Treatments with proteinase K, trypsin and chymotrypsin altered the protein structures such that the reactivity o~ the MAbs Sp-5 and Sp-8 ~ .

in Western immunoblot was completely abolished. Sodium periodate oxidation did not produce significant alteration in the reactivity of the MAbs with their epitopes.
Reactivity_of MAbs Against a Panel of S. Pneumoniae Isolates Dot enzyme immunoassay and immunoblot analysis were performed to determine the reactivity of the MAbs against a panel of 128 S. pneumoniae strains representing 30 capsular serotypss. The frequency of strains reactive with the individual MAbs was 100% for MAb Sp-5 and 100% for MAb Sp-8 (see Table 2).

Properties of MAbs Directed Aqainst Pneumococcal Proteins Reactivity with Bacterial St_ains*
MAb Iq** Specificity Pneumococcus Non-pneumococcus -Sp-5 IgGl, 40-kDa protein 128tl28 0/83 Sp-8 IgGl, 67-kDa protein 128/128 1/83***
* Reactivity was tested by immunodot and Western immunoblot assays.
** Immunoglobulin class, subclass and light chain.
*** One S. sanquis isolate was recognized.

When tested by dot enzyme immunoassay, 120 and 125 out of 128 pneumococcal strains were recognized by MAb Sp-5 and MAb Sp-8, respectively. Western immunoblotting analysis of strains unreactive by dot immunoassay indicated that both proteins, the 40-kDa and the 67-kDa, were present on all pneumococcal isolates since all strains were detected using the latter assay. Eighty three different non-S. pneumoniae strains were used to study the specificity of the MAbs. These strains represented 22 genera and 44 species of bacteria. With the exception of one S. sanguis isolate which reacted with MAb Sp-8, none of the non pneumococcal strains reacted wi~h the MAbs when tested by either dot enzyme or Western immunoblot assay.
Surface Accessibility of Sp-5 and Sp-8 Epitoees To determine if the MAbs Sp-5 and Sp-8 was directed against cell surface-exposed epitopes, hybridoma culture supernatants were incubated with intact live bacteria. The binding of MAbs to the bacterial surface was then analyzed by a radioimmunoassay using 125iodine-labelled anti-mouse immunoglobulins. Nonspecific binding was recorded using a control IgGl MAb. The results indicated that Sp-5 and Sp-8 MAbs bound to surface-exposed epitopes on S. pneumoniae strains. The MAbs did not bind to the surface of the serotype 8 S. suis strain C-35.
Summary Although S. pneumoniae remains an important human pathogen, diagnosis of pneumoccoccal disease continues to be a problem. A sensitive and specific method for the rapid detection of a current S. pneumonlae infection is highly desirable. Limitations associated to cultivation of clinical samples have led to the development of immunoassays for the detection of pneumococcal antigens. The use of highly 21~ / ~5l~5 specific MAbs lacking batch-to-batch variations that may easily be obtained in large amounts offers many advantages over the use of polysera. The present invention provides hybrid cell lines which produce MAbs against S. pneumoniae proteins.
Pneumococcal proteins with molecular masses of 40 and 67-kDa were reactive with MAbs identified as Sp-5 and Sp-~, respectively, as demonstrated by SDS-PA~E followed by immunoblot analysis. Further evidence that the MAbs were directed against protein epitopes were obtained when they failed to rea~t with S. pneumoniae antigens treated with proteolytic enzymes. Binding radioimmunoassay with live bacteria clearly showed that the proteins were cell surface-exposed. Since the pneumococcal strains selected for this assay included the most prevalent capsular serotypes, the tests provide strong evidence that both proteins are broadly expressed at the bacterial cell surface among clinically important serotypes of this bacterium. It is also clear that the presence of a capsule does not prevent the binding of the MAbs to the surface of the bacteria. The binding of MAbs differed considerably from one strain to the other indicating that the amount of 40 and 67-kDa proteins and/or the protein exposure to the bacterial cell surface may vary. This observation provides a possible explanation to the discrepancies observed between dot and immunoblot MAb reactivities. Interestingly, the level of binding was ' constant from one experiment to the other and is likely a strain characteristic.
Numerous applications of MAbs have been described.
They may be used for direct detection of antigen in clinical samples if they have two major qualities - specificity and sensitivity. Both MAbs recognized epitopes that are highly conserved among S. pneumoniae strains since all strains tested were shown to produce the proteins. In immunoblot analysis, bands of the same molecular masses were found in all cases regardless of the capsular serotypes and the nature (whole bacteria versus semi-purified proteins) of the antigenic preparations. Similarly, the lack of variation in the molecular masses of the 40 and 67-kDa proteins might suggest a high degree of antigenic conservation of the two pneumococcal proteins.
The results confirmed the high cpecificity of MAbs Sp-S and Sp-8 for S. pneumoniae. MAb Sp-5 did not show immunological cross-reactivity with closely related species or with other gram positive and gram negative bacteria. MAb Sp-8 reacted with one out of eighty-three non-pneumococcal strains indicating that its specific epitope is rarely borne by bacteria other than S. pneumoniae.
The surface of bacterial cell presents a diverse menu of potential antigens for vaccine and diagnosis development. It has been shown that two S. pneumoniae proteins constitute common antigen among isolates of this 2~?~ k58~
.
bacterium. The Sp-5 and Sp-8 MAbs based-immunoassays for direct detection of antigen in clinical samples should be able to detect soluble antigens released from the bacteria as well as to detect living or dead bacteria since positive reactions were obtained in dot and accessibility assay using whole bacteria and ELISA and immunoblo~ using soluble proteins.

Claims (10)

1. A monoclonal antibody which specifically reacts with surface accessible protein antigens of the bacterium Streptococcus pneumoniae.

2. A monoclonal antibody according to claim 1, which reacts specifically with a protein having a molecular mass of 40-kDa.

3. A monoclonal antibody according to claim 1, which reacts specifically with a protein having a molecular mass of 67-kDa.

4. The hybridoma cell line which produces a monoclonal antibody specifically reactive with surface accessible proteins antigens of the bacterium Streptococcus pneumoniae.

5. The hybridoma cell line of claim 4, which produces a monoclonal antibody specifically reactive with a protein having a molecular mass of 40-kDa.

6. The hybridoma cell line of claim 4, which produces a monoclonal antibody specifically reactive with a protein having a molecular mass of 67-kDa.

7. The hybridoma cell line of claim 5, formed by immunizing mice with Streptococcus pneumoniae antigens; and fusing immunized spleen cells from the mice with SP2 myeloma cells.

8. The hybridoma cell line of claim 6, formed by immunizing mice with Streptococcus pneumoniae antigens; and fusing immunized spleen cells from the mice with P3-X63-Ag 8.653 myeloma cells.

9. A hybridoma cell line consisting essentially of hybridoma clone Sp-5.

10. A hybridoma cell line consisting essentially of hybridoma clone Sp-8.