Monoclonal anti-body to cell surface protein of the bacterium Streptococcus pneumoniae

This invention relates to a monoclonal antibody (MAb) directed against a surface protein of Streptococcus pneumoniae, a hybridoma cell line producing said antibody, and the use of such an antibody to detect the bacterium Streptococcus pneumoniae, or to detect antigens of Streptococcus pneumoniae.

BACKGROUND OF THE INVENTION 
The present invention involves a monoclonal antibody (MAb) with the 
specificity for a 67,000 dalton cell surface protein of Streptococcus 
pneumoniae, a cell line that produces said antibody, and the partially 
purified 67,000 dalton cell surface protein. 
S.pneumoniae is the leading cause of community-acquired bacterial pneumonia 
(pneumococcal disease) with approximately 500,000 cases a year reported in 
the United States. Bacterial pneumonia is the most prevalent among the 
very young, the elderly and immuno-compromised persons. In infants and 
children, pneumococci are the most common bacterial cause of pneumonia, 
otitis media and bacteremia and a less common cause of meningitis (causing 
20-25% of reported cases). 
Pneumococci are carried in the respiratory tract of a significant number of 
healthy individuals. In spite of the high carriage rate, its presence does 
not necessarily imply infection. However, if one of the highly pathogenic 
pneumococcal types, such as S.pneumoniae, is isolated from rusty-coloured 
sputum (also containing a large number of polymorphonuclear leucocytes), 
body fluids, blood cultures, or specimens collected via transtracheal or 
lung puncture from the lower respiratory tract, its detection is usually 
significant. 
Detection of this bacteria at an early stage is essential to facilitate 
treatment of the infection. Thus, it is important to possess the ability 
to identify whether S.pneumoniae is present in a patient and to be able to 
follow the effect of antibiotic treatment on the bacteria. As available 
immunoassays for S.pneumoniae antigen detection have shown lack of 
specificity and/or sensitivity, there remains the need for an improved 
method of such detection. 
S.pneumoniae is a gram positive bacteria. Proteins located on the cell 
surface of many gram positive bacteria have, in the past, been used in 
typing and immunoprotection studies. There are a large number of 
S.pneumoniae strains, and there are many cell surface proteins associated 
with S.pneumoniae. This has made the identification of a common but 
exclusive cell surface antigen difficult. However, MAb technology has 
provided researchers with tools to reproducible and accurately analyze the 
cell surface components of S.pneumoniae. In addition, S.pneumoniae 
proteins are of interest to epidemiologists as they may provide for 
vaccines against the bacteria. 
One such pneumonococcal capsular polysaccharide vaccine has been developed, 
which incorporates the polysaccharide antigen of 23 serotypes of 
pneumococci that are responsible for 87% of pneumococcal disease in the 
United States. This second generation vaccine replaced the 14-valent 
polysaccharide vaccine licensed in 1977. However, the U.S. Department of 
Health and Human Services states that a more immunogenic pneumococcal 
vaccine is needed, particularly for children younger than 2 years of age. 
This is because the 23-valent vaccine is poorly antigenic in this age 
group, and its use is not recommended in children with recurrent upper 
respiratory diseases, such as otitis media and sinusitis. Furthermore, the 
23-valent vaccine is only 44-61% efficacious when administered to persons 
over 65 years old, and revaccination is not advised. Thus, there remains 
the need for an improved pneumococcal vaccine. 
It follows then, that there remains a need for at least two products 
relating to S.pneumoniae. The first is a rapid, specific, and sensitive 
diagnostic technique for of all strains of S.pneumoniae, that does not 
give false positive results. What is optimally desired is a Mab that will 
recognize a cell surface antigen that is universally present in most, if 
not all, strains of S.pneumoniae and, at the same time does not recognize 
other organisms or material which may be found in conjunction with 
S.pneumoniae. Secondly, it is desirous that the Mab and said 67,000 dalton 
protein be used in research towards development of an improved vaccine. 
SUMMARY OF INVENTION 
The present invention involves a Mab that is reactive with an epitope (an 
antigenic determinant of known structure) of a proteinaceous surface 
component of the bacterium S.pneumoniae, with said antibody being reactive 
with said antigen in at least 96% of strains of S.pneumoniae. 
It is preferred that such MAb is reactive with an epitope of a 
proteinaceous cell surface component of the bacterium S.pneumoniae, 
particularly a protein of approximately 67,000 daltons. 
An additional aspect of this invention involves a cell line capable of 
producing a MAb that is reactive with an epitope of a proteinaceous cell 
surface component of the bacterium S.pneumoniae, with said epitope being 
present in at least 96% of strains of said bacterium. 
It is preferred that said cell line be capable of generating a MAb that 
demonstrates specificity for an epitope of a proteinaceous cell surface 
component of the bacterium S.pneumoniae. It is preferred that said cell 
line is a hybridoma cell line, specifically a hybrid of a mouse spleen 
cell and an immortal myeloma cell. 
A further aspect of this invention provides a diagnostic method to 
identify, type, and/or detect the presence of the bacterium S.pneumoniae 
or its antigens, with such method (a) causing the test sample to come into 
contact with said MAb; and (b) observing whether cell-labelling or 
agglutination occurs, indicating the presence of S.pneumoniae or an 
antigen of S.pneumoniae. 
It is preferred that such a method involves a MAb that is reactive with an 
epitope of a proteinaceous cell surface component that is present in at 
least 96% of the known strains of S.pneumoniae. It is additionally 
preferred that the said label is chosen from a radio-label, a fluorescent 
label, a colloidal gold label, and a biotin label or an enzyme label. This 
method could also be employed to detect infection of S.pneumoniae in 
patients. 
An additional feature of this invention provides a significantly purified 
form of the said proteinaceous cell surface component of the bacterium 
S.pneumoniae, having an epitope present in at least 96% of strains of said 
bacterium. A preferred embodiment of this feature is a 67,000 dalton 
protein or fragment thereof containing such an epitope. It is to be 
preferred that an epitope of said component or part thereof is present in 
more than 99% of the strains of S.pneumoniae, and is only present in said 
bacterium. 
I have generated a MAb that specifically recognized an epitope of a 
proteinacous cell surface component of the S.pneumoniae common to more 
than 96% of all strains of said bacterium. The use of this MAb for 
immunodiagnosis and typing is disclosed.

DETAILED DESCRIPTION OF THE INVENTION 
The production of a monoclonal antibody directed against a common protein 
of S.pneumoniae. 
The strains of bacteria and culture conditions 
S.pneumoniae strains were obtained from clinical isolates from Children's 
Hospital of Eastern Ontario, Ottawa, Laboratoire de la Sante Publique de 
Quebec, Sainte-Anne de Bellevue, and Trinidad. S.pneumoniae was grown on 
chocolate agar plates supplemented with 1% ISOVITALEX.RTM. (BBL, 
Cockeysville, Md.) overnight at 37.degree. C., in an atmosphere containing 
5% CO.sub.2. The resulting cultures were stored in 
brain heart infusion broth containing 20% glycerol at -70.degree. C. 
Protein preparation 
The extraction of the proteins from the bacteria was performed using 
SABCOSYL. Whole cells (from 50 plates) suspended in phosphate buffered 
saline PBS (45 ml) were heat-killed at 56.degree. C. for 20 minutes and 
centrifuged at 3600 RPM Sorvall SS-34 rotor with Rmax=10.70 cm for 30 
minutes using a fixed angle. The pellet was resuspended in 14 ml of 10 mM 
Hepes buffered water, pH7.4. The cells were then sonicated using a Vibra 
Cell sonicator, 4 times .times.30 seconds pulse at 50%, 30 seconds between 
each sonication. The suspension was centrifuged at 3500 RPM in a Sorvall 
SS-34 rotor with Rmax=10.70 cm for 20 minutes. The supernatant was 
centrifuged once more to get it more clear. It was then transferred to a 
rigid wall polycarbonate tube, and ultracentrifuged at 28.8K (28,800 RPM) 
for 60 minutes, 7.degree. C. using a 50.2 Ti rotor. The supernatant was 
discarded and the pellet resuspended in 1 ml of 10 mM Hepes buffered 
water. Fifteen seconds of sonication was necessary to resuspended the 
pellet. One ml of 2% N-Lauryl Sarcosine (SARCOSYL) in 10 mM hepes buffered 
water was added to the suspension and the tube was gently shaken for 
approximately 3 minutes at room temperature to mix. The suspension was 
ultracentrifuged again, at 28.8 K for 60 minutes at 7.degree. C. The 
protein content of the supernatant was determined by the BIO-RAD protein 
assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada). 
Immunization of mice 
A BALB/c mouse was inoculated sub-cutaneously with 5 .mu.g of S.pneumoniae 
strain Trinidad 810062 proteins from the sarcosyl extraction, combined 
with 25 .mu.g of Quil A. Three weeks later, the mouse was reinjected 
subcutaneously with 5 .mu.g proteins and 25 .mu.g Quil A. Eight days 
before the hybridoma production, the mouse was given 5 .mu.g proteins and 
25 .mu.g Quil A, sub-cutaneously. Six and three days before the fusion, 
the mouse received 5 .mu.g of the same protein preparation, but without 
Quil A, and the injection was done intraperitoneally. Serum was obtained 
from the immunized mouse by cardiac puncture before spleen removal. 
Fusion procedure 
Hybridomas were produced according to a modification of the methods 
described by Fazekas De St. Groth and Scheidegger, J. Immunol Methods, 
vol. 35, 1-21 (1986). Spleen cells from immunized mouse and nonsecreting, 
HGPRT deficient, mouse myeloma cells P3.times.63 Ag 8.653 were fused in a 
ratio 10:1 in Dulbecco modified Eagle's medium (DMEM, Flow Laboratories, 
Mississauga, Ontario, Canada) containing 50% (w/v) polyethylene glycol 
1000 (T.J. Baker Chemical Co., Phillipsburg, N.J.). The fused cells (0.1 
ml, 1.5.times.10.sup.5 cells/ml) were portioned into 96-well tissue 
culture plates (Costar plastics, Vineland, N.J.) which contained a feeder 
layer of 4.times.10.sup.3 murine peritoneal exudate cells (macrophages). 
The suspensions of cells were grown in DMEM that were supplemented with 
20% bovine calf serum (Gibco), 2 mM L-glutamine (Sigma Chemical Co., St. 
Louis, Mo.), and 50 .mu.g/ml gentamicin (Sigma) in the presence of 
hypoxanthine, aminopterin, and thymidine (HAT) selection medium. All 
cultures were checked on day six for the presence of clones and the medium 
was changed. Supernatants of wells containing growing clones were tested 
on day twelve by ELISA for MAb directed against S.pneumoniae antigens. The 
cells that were producing antibody were subcloned through limiting 
dilution. Subclones that were selected were grown either as ascites 
according to the method of Brodeur et al, J. Immunol Methods, 71, 265-272 
(1984) or in vitro for freezing in liquid nitrogen. 
Immunoglobulin class determination 
The supernatant from the cells producing antibodies were tested against 
affinity purified anti-mouse immunoglobulin (Southern Biotech) using the 
ELISA method. 
Enzyme-Linked Immunosorbent Assay (ELISA) Procedure 
Screening of resulting supernatants for MAbs directed against S.pneumoniae 
was performed as described by Brodeur et al, J. Med. Microbiol, vol. 15, 
1-9, (1982). The antigen (0.1 ml) containing 0.75 .mu.g protein in 0.05M 
carbonate buffer at pH 9.6 was portioned into each well of a High-binding 
microtiter plate (Flow). The plate was incubated overnight at room 
temperature to permit the adsorption of the antigen. The plate was then 
washed with PBS containing 0.02% Tween-20 (Sigma) and 150 .mu.l of 0.5% 
bovine serum albumin (BSA, Sigma) in PBS was added to each well. The plate 
was incubated at 37.degree. C. for 30 minutes. The BSA was discarded and 
the plate was washed and the test supernatants were added. The positive 
control was a standard serum. After a one hour incubation at 37.degree. 
C., the plate was washed three times. This was followed with the addition 
of 0.1 ml alkaline phosphatase-conjugated goat anti-mouse immunoglobulins 
(Miles Laboratories, Elkart, Ind.) diluted 1:1000 in PBS containing 3% 
BSA. The plate was incubated at 37.degree. C. for an additional 1 hour. 
The plate was then washed and 0.1 ml of a 10% diethanolamine solution (pH 
9.8), containing 1 mg/ml p-nitrophenylphosphate (Sigma) was added. The 
plate was allowed to stand for sixty minutes. The absorbance was then 
determined spectrophotometrically using a DYNATECH.RTM. microplate reader 
MR 600 at 410nm. Readings greater than 0.1 were scored as positive, 
indicating the presence of antibodies directed against S.pneumoniae. 
SDS-polyacrylamide gel electrophoresis (PAGE) 
Resolution of proteins was achieved through electrophoresis on sodium 
dodecyl sulfate (SDS) 0.75 mm thick slab mini gels according to the method 
described by Laemmli, Nature, vol. 227, 680-685 (1970). A 10% acrylamide 
(Bio-Rad) resolving gel and a 4.0% stacking gel were utilized. Cell 
lysates used on the gels were prepared by sonication, SARCOSYL extraction 
or heat-killed whole cell preparation. Lysates were mixed with sample 
buffer (62.5 mM Tris-HCl) pH 6.8, 1% (v/v) glycerol, 2% (w/v) SDS, 0.5% 
(v/v) 2-mercaptoethanol and 0.5% (w/v) bromophenol blue) and heated for 5 
min. at 100.degree. C. Aliquots of 15 .mu.l containing 7.5 .mu.g of 
protein were applied to each gel lane. Electrophoresis was carried out at 
100 V constant voltage until the bromophenol blue tracking dye entered the 
separating gel. At this time, the voltage was then increased to 200 V. The 
gels were stained with Coomassie blue dye and then destained following the 
method of Weber and Osborn in J.Biol. Chem. vol. 244, 4406-4412 (1969). 
The protein standards used were: Phosphorylase b (97,000), Bovine serum 
albumin (66,200), ovalbumin (45,000), carbonic anhydrase (28,000), Soybean 
Trypsine Inhibitor (20,100), .alpha.-lactalbumin (14,200) (Bio-Rad 
Laboratories, Mississauga, Ontario, Canada). 
Immunoblotting procedure 
The proteins were transferred electrophoretically from the SDS-PAGE gel to 
nitrocellulose paper (Bio-Rad) by the method described by Towbin et al., 
Proc. Nati. Acad. Sci., vol. 76, 4350-4354 (1979). A constant current of 
66 mA was applied to the gel-nitrocellulose paper sandwich for 15 minutes. 
This was done in an electroblot buffer of 25 mM Tris-HCl, 192 mM glycine 
and 20% (v/v) methanol at pH 8.1. The proteins transferred onto the blot 
were either stained with amido black or detected by an enzyme immunoassay. 
The detection of bacterial antigens was performed by soaking the paper in 
PBS solution containing 1% milk for 30 minutes in order to block 
non-specific protein binding sites. The paper was then incubated with 
mouse hyper-immune sera at 37.degree. C. for 1 hour. The sheet was washed 
three times with PBS followed by a 1 hour incubation at 37.degree. C. with 
peroxidase-conjugated goat anti-mouse immunoglobulins (Cappel, 
Cochranville, Pa.) diluted 1:1000 in PBS containing 3% BSA. The sheet was 
once again washed three times and the blots were soaked in a solution of 
o-dianisidine prepared as described by Towbin et al (supra). 
Dot-enzyme immunoassay 
A dot-enzyme immunoassay was used for a quick method of screening several 
MAbs against a large number of S.pneumoniae strains. The strains were 
grown on chocolate agar plates overnight and an aliquot of approximately 
3.times.10.sup.9 bacteria/ml was prepared in PBS. A small amount of the 
suspension, approximately 40 .mu.l was applied to a nitrocellulose paper 
using a DOT-BLOT apparatus (Bio-Rad Laboratories, Mississauga, Ontario, 
Canada). The dot nitrocellulose paper was then processed following the 
procedure described in the immunoblotting procedure. 
Enzymatic treatment of proteins 
Nitrocellulose paper with transferred proteins (see immunoblotting 
procedure) was treated with 3 different enzymes before being processed 
with the MAb. The paper was soaked in a 1.25 ,mg/ml Proteinase K solution 
for 1/2 hour, a 150 .mu.g/ml Trypsin solution for 2 hours, or in a 1 mg/ml 
Chymotrypsin solution for 2 hours. The nitrocellulose paper was then 
processed with the MAb as described in the immunoblotting procedure. These 
treated papers were observed for the disappearance of the protein band. 
The normal immunoblot, without enzymatic treatment, was used as a positive 
control. 
Properties of monoclonal antibodies 
More than 450 hybrid clones were obtained by fusing sensitized mouse spleen 
cells with P.sub.3 .times.63 Ag8.653 cells. The screening for the MAbs in 
the hybridoma culture supernatants was performed by ELISA, utilizing the 
homologous immunizing S.pneumoniae SARCOSYL extract as the coating 
antigens. Every positive hybrid clone supernatant was further tested 
against several other strains of S.pneumoniae. Eight hybridoma cell lines 
that demonstrated different patterns of reactivity in ELISA were obtained 
(see Table 1). 
TABLE 1 
__________________________________________________________________________ 
Characterization of monoclonal antibodies directed against 
S. pneumoniae antigens. 
Immunoglobulin 
O.D. at 
Antigen Specificity 
Clone Class/subclass 
410 mm 
recognized to S. pneumoniae 
__________________________________________________________________________ 
1) 
1G-4 
IgG.sub.1 
0.154 
protein, few strains only 
approximately 
72 KDa 
2) 
2D-4 
IgM &gt;2.000 
carbohydrate 
non-specific 
3) 
2G-1 
IgM 0.136 
N/A N/A 
4) 
4A-9 
IgG.sub.3 
&gt;2.000 
carbohydrate 
non-specific 
5) 
6B-5 
IgA 0.294 
carbohydrate 
non-specific 
6) 
6E-9 
IgM/IgG.sub.1 
1.000 
proteins, non-specific 
approximately 
67 KDa and 100 KDa 
carbohydrate 
7) 
11E-1 
IgG1 0.124 
protein, yes 
approximately 
67 KDa 
8) 
13H-8 
IgG2A 0.364 
protein, homol. strain 
approximately 
only 
72 KDa 
__________________________________________________________________________ 
MAb 11E-1 was the only clone that was very specific to all the strains of 
S.pneumoniae. It was also directed against a protein. This MAb was 
subcloned twice by limiting dilution and the class and subclass were 
determined using affinity purified anti-mouse immunoglobulikn (Southern 
Biotech) in an ELISA test. This clone was then identified as 11E-1H-3/F-11 
but 11E-1 kept as the official designation. 
Clone 11E-1 was deposited with the American Type Culture Collection, 12301 
Parklawn Dr., Rockville, Md. on Feb. 4, 1993, under the ATCC accession 
number HB 11262. 
Identification of antibody-specific epitopes on the antigen 
The Western immunoblotting technique was used to ascertain the specific 
antigen to which each MAb binds. The mouse hyperimmune serum that was used 
as positive control, detected all the major proteins present in strains of 
S.pneumoniae. 
Seven of the eight MAbs reacted with antigens transferred from the SDS-PAGE 
to nitrocellulose paper. The remaining MAb was too weak to react. Three 
different proteins were recognized by the MAbs with apparent molecular 
weights of 100,000, 72,000 and 67,000 daltons. In addition a number of 
very low molecular weight carbohydrates were recognized. 
Binding properties of monoclonal antibody 11E-1 
To determine whether clone 11E-1 was directed against the cell surface 
exposed epitope of the 67,000 dalton protein, or part thereof, hybridoma 
culture supernatants containing the MAbs were incubated with live intact 
S.pneumoniae bacterial cells. The bacteria were then washed twice with PBS 
and incubated with .sup.125 I-labelled goat anti-mouse Ig (Dupont) and 
pelleted. 
The bacterial cell-bound .sup.125 I was counted using a 1282 Compugamma. 
Fewer than 3000 cpm were obtained using negative controls. These data 
represent the mean of triplicate determinations. 
Supernatant containing the MAb 11E-1 showed counts between 5 to 9 times the 
negative controls containing no MAb, indicating that the component is 
surface accessible. 
Specificity of monoclonal antibody 11E-1 
The initial ELISA characterization showed 11E1 reacted only with 
S.pneumoniae strains. A dot-enzyme immunoassay was used for a rapid method 
of screening this MAb against numerous bacterial strains. The MAb 11E-1 
reacted specifically with 118 S.pneumoniae strains and only cross reacted 
with one strain of Streptococcus sanguis type I (Table II) 
TABLE II 
______________________________________ 
Specificity of monoclonal antibody 11E-1 
Bacterial strains 
Reactivity by DOT-blot.sup.1 
______________________________________ 
S. pneumoniae 118/123 
other Streptococcus sp. 
.sup. 1/29.sup.2 
N. meningitidis 0/8 
other Neisseria sp. 
0/7 
E. coli 0/7 
S. aureus 0/1 
H. influenzae 0/1 
K. pneumoniae 0/1 
S. epidermidis 0/2 
______________________________________ 
.sup.1 Number of positive/Number of Strains 
.sup.2 Positive strain is S. sanquis I ID 12315 from LSPQ, SteAnne de 
Bellevue, Quebec Note: Of the 5 strains of S.pneumoniae that are not 
recognized by DOT-assay, 4 have been tested by immunoblot indicating that 
the 67KDa. 
Preparation of Protein Antigen Extract 
Several preparative methods of protein extracts have been utilized, 
especially for SDS-PAGE gel electrophoresis. The sarcosyl extraction has 
been described previously. An additional method involved the sonication of 
the bacteria. Approximately 10" bacteria were suspended in 5ml PBS and 
heat-killed for 20 minutes at 56.degree. C. Using a SONIFIER CELL 
DISRUPTOR 350, (pulse was set at 50%), the cells were sonicated 3.times.5 
minutes, being kept on ice during the entire procedure. The suspension was 
the centrifuged for 20 minutes at 25000 RPM using a 70 Til rotor run at 
10.degree. C. The supernatant was kept and the protein content determined 
by the BIO-RAD protein assay. Whole cell extract was also used, 50 .mu.l 
of 10% SDS was added to the bacterial suspension, which was then boiled 
for 20 minutes and centrifuged.