Note: Descriptions are shown in the official language in which they were submitted.
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
TABLE 1
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 &
---- ' :
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.
2~
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).
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.
16
