Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02770771 2012-02-10
MONOCLONAL ANTIBODIES FOR PBP2-a PROTEIN AND HOMOLOGOUS
SEQUENCES FOR TREATMENT OF INFECTIONS AND
IMMUNODIAGNOSIS ON BACTERIA FROM PHYLUM FIRMICUTES
Invention Field
The current invention refers to monoclonal antibodies able to recognize and
bind to
PBP2a protein and other proteins presenting sequences homologous to PBP2a,
including the pathogens methicillin-resistant Staphylococcus aureus - MRSA,
coagulase-negative Staphylococcus, Staphylococcus sciuri, Enterococcus spp.,
and any
other bacterium possessing PBP2a or sequences homologous to this protein.
The invention still refers to the use of monoclonal antibodies able to
recognize and
bind to PBP2a protein and other proteins presenting sequences homologous to
PBP2a in
a complementary immunodiagnosis for detection of resistance to beta-lactams.
Invention Rationales
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a
major
concern cause for clinicians, presenting mortality and morbidity rates higher
than the
ones of infections caused by methicillin-sensitive Staphylococci (1).
Furthermore, these
infections cause longer hospitalization time and higher expense with
antimicrobials,
leading to a higher cost in treatment of patients infected by this pathogen
(1).
Vancomycin has been the first choice antimicrobial for treatment of infections
caused
by MRSA. However, the growing isolation of MRSA strains in communities in the
United States and Australia (2, 3, 4), together with the identification of
MRSA strains
with intermediate resistance to vancomycin in Japan, United States (5), and
Brazil (6),
cause the current scene to become more severe. The description of MRSA strains
totally
resistant to vancomycin in 2004 (7) has caused huge concern in the medical-
scientific
community. MRSA now represents a strong candidate for becoming the fearful
"superbug or superbacterium" - a pathogen resistant to all drugs available
nowadays.
Usually, MRSA prevalence rates (ratio of all infections by S. aureus caused by
MRSA) in hospital infections are gradually increasing in the last decades. In
a study
conducted by Jarvis et al., including 1268 ICUs (intensive care units) in 337
hospitals in
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the United States, the number of infections by MRSA in ICUs changed from 660
to
2184 cases and the prevalence increased from 35% to 64,4% (8). In Japan,
prevalence
rates of hospital infections (HIs) caused by MRSA can present worrying values,
from
60% to 90% (9). In studies conducted in the United States, the percentile
changed from
2% in 1974 to 50% in 1997 (10, 11) and, in some hospitals in the United
States, more
than 80% of HIs are caused by MRSA (12). In England, the prevalence increased
from
1.5% to 15.2% between 1989 and 1995 and now (2004) the estimate is 41.5% (13).
Besides high prevalence rates, especially in teaching and large-sized
hospitals,
MRSA is deemed as the main pathogen causing epidemic outbreaks in Brazilian
hospitals (14). In 1986, more than 50% of S. aureus strains with hospital
origin isolated
in patients from university hospitals in Sao Paulo were resistant to
methicillin and, in
1993, the incidence of MRSA in the Pediatric Hospital of Paulista Medicine
School
was 70% (15). In a study conducted in hospitals in Belo Horizonte, Resende et
al. (16)
pointed out a prevalence of 71% of MRSA.
MRSA strains present a penicillin-binding protein with very low affinity for
antimicrobials in the beta-lactam class, such as PBP2a (17). In the presence
of this
enzyme, which is codified by gene mecA, the bacterium is successful to
synthesize the
peptidoglycan, even in the presence of beta-lactams. This enzyme can be also
found in
coagulase-negative Staphylococcus and in Staphylococcus sciuri - a bacterium
present
in the normal flora of dogs. Besides resistance to beta-lactams, hospital MRSA
strains
present resistance to most other available antimicrobial classes, with the use
of
glycopeptides (vancomycin and teicoplanin) remaining as first choice
treatment.
Two studies using DNA vaccine against PBP2a showed that this protein is
immunogenic and that the obtained immune response was able to confer
protection
against MRSA in assays conducted in murine model (18, 19). However, it is
known
that, in hospital infections, most patients are immunodepressed (20). In this
cases, a
vaccine would not be able to generate protective antibodies in due time to
control a
bacterial infection.
PBP2a Immunogenicity
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PBP2a is a class II multimodular enzyme according to Goffin and Ghuysen's
classification (40). This 76-kilodalton enzyme is composed by a membrane-
binding
region, a non-transpeptidase domain, and a transpeptidase domain, containing a
4-amino
acid active core (STQK), responsible for the bacterial transpeptidation
reactions (20 bis
Ryfell, 1990).
State of the art studies with the DNA vaccine against PBP2a show that the
results of
bacterial reduction (renal quantification) in immunized animals subjected to
challenge
by systemic infection were of 3 to 4 times in the work of Ohwada et al. and of
1000
times in the work of Senna et al. The authors of these studies used the full
sequence
(except the membrane fixation region) of the gene mecA and an internal
fragment of
transpeptidase domain, respectively.
But, according to what was mentioned above, a vaccine is not able to generate
protective antibodies in due time to control a bacterial infection. Thus, in
case of
infections by MRSA, the administration of anti-PBP2a monoclonal antibodies is
the
most proper therapy for treating these infections.
Invention Summary
The main objective of the current invention is to provide monoclonal
antibodies able
to recognize and to bind to PBP2a protein (SEQ ID NO.:1) and other proteins
presenting sequences homologous to PBP2a, including the pathogens methicillin-
resistant Staphylococcus aureus - MRSA, coagulase-negative Staphylococcus,
Staphylococcus sciuri, Enterococcus spp., and any other bacterium possessing
PBP2a or
sequences homologous to this protein.
Another invention objective is the use of monoclonal antibodies able to
recognize and
bind to the PBP2a protein and other proteins presenting sequences homologous
to
PBP2a in a complementary immunodiagnosis for detection of resistance to beta-
lactams.
The monoclonal antibodies of the current invention are represented by
sequences
SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10,
SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.:
15, SEQ ID NO.: 16, and SEQ ID NO.: 17.
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Brief Figure Description
Figure I shows the immunoenzyme test (ELISA) of sera of animals immunized for
producing anti-PBP2a antibodies.
Figure 2 shows the result of a polyacrylamide gel with raw samples and samples
after
purification.
Figure 3 shows the immunoblotting test of MRSA and MSSA lysates against a
supernatant containing anti-PBP2a monoclonal antibodies.
Figure 4 is a representation of flow cytometry results of MRSA (CEB) and MSSA
bacteria, incubated with anti-PBP2a monoclonal antibody and marked with
phycoerythrin (PE).
Figure 5 shows the in vitro protection test (MIC - minimum inhibitory
concentration)
conferred by anti-PBP2a purified monoclonal antibody and vancomycin against an
inoculum of different MRSA strains.
Figure 6A shows renal quantification results in treated and non-treated
animals with
the anti-PBP2a monoclonal antibody, subjected to systemic infection with a
sublethal
dose of MRSA CEB strain.
Figure 6B shows renal quantification results in treated and non-treated
animals with
the anti-PBP2a monoclonal antibody, subjected to systemic infection with a
sublethal
dose of MRSA Iberian strain (European epidemic clone).
Figure 6C shows renal quantification results in treated and non-treated
animals with
the anti-PBP2a monoclonal antibody, subjected to systemic infection with a
sublethal
dose of WB79 CA-MRSA strain (Brazilian community strain).
Figure 7A shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a lethal bacterial dose (MRSA CEB).
Figure 7B shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a lethal bacterial dose (Iberian MRSA).
Figure 7C shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a lethal bacterial dose (WB79 CA-MRSA).
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Figure 8A shows the bacterial quantification in kidneys of animals treated
with anti-
PBP2a monoclonal antibody, vancomycin, and antibody + vancomycin association
and
non-treated animals after infection with bacteria (MRSA CEB).
Figure 8B shows the bacterial quantification in kidneys of animals treated
with anti-
PBP2a monoclonal antibody.
Figures 9 show the interaction between recombining PBP2a (antigen) and
monoclonal antibodies clone 38 (Figure 9A) and clone 10 (Figure 9B).
Figure 10 is a graph of the second analysis by flow cytometry of MRSA samples
in
presence of FITC-marked anti-PBP2a antibody.
Figure 11 shows the result of the in vitro protection conferred by the
antibody against
VRE enterococcus strain.
Figure 12 refers to LD50 and lethal dose for intraperitoneal infection
determination.
Figure 13 shows the efficacy result of in vivo protection of anti-PBP2a and
PBP5
monoclonal antibody from enterococci against systemic infection.
Figure 14 shows renal quantification results of the in vivo protection test ¨
systemic infection by
intraperitoneal route in murine model with vancomycin-resistant Enterococcus
faecium.
Detailed Invention Description
The emergence of infections by bacteria multiresistant to antimicrobials is
presenting
an alarming increase. The prevalence of hospital infections caused by MRSA has
increased in all world parts present high morbidity and cause high cost due to
the
intensive use of antimicrobials and to the longer period of patient
hospitalization (24).
The treatment based on chemotherapy has been showing signs of exhaustion, with
appearance of very few really novel and efficient drugs in the market (25).
In this context, passive immunotherapy (re)appears as a promising alternative,
presenting a range of advantageous features compared to conventional
chemotherapy:
among them, the lower toxicity, higher plasma half-life (which facilitates the
treatment
by dose reduction and maybe a lower final treatment cost), and, especially in
the case of
the product presented here, selective toxicity, recommended by Paul Ehrlich
(where the
drug selectively eliminates the referred pathogen) (25a). This last feature is
extremely
useful to prevent the so called selection pressure, played by broad-spectrum
antimicrobials - which provides the appearance of multiresistant bacteria
(26).
CA 02770771 2012-02-10
In order to develop this product, the inventors selected PBP2a as target,
based on
preliminary studies with DNA vaccine in murine models (18, 19, 21) and
immunostructural molecule analyses. PBP2a had its structure elucidated in 2002
(29),
and is deposited at PDB (Protein Data Bank) under the code lwmr.
Differently from other approaches, the inventors worked with an internal
region of
the molecule with 76 amino acids, which flank the enzyme active core (SxxK),
at the
transpeptidase domain. Epitope identification analyses were performed at the
molecule
and in their presentation to the different class II CHM alleles (Tepitope
program), with
later verification of their location at the molecule (SPDB-Viewer program).
This
approach enables us to evaluate the facility of antibody access to these
targets at the
native molecule.
This computerized analysis showed the presence of epitopes close to the enzyme
active core, with excellent recognition degree by class II CHM alleles,
located at PBP2a
surface (not shown data).
These in silico previews were confirmed by the performed target recognition
tests
(immunoblotting and flow cytometry), and the antibody binding to this PBP2a
region
showed to be able to confer high protection, demonstrated by the results
obtained in the
performed in vitro and in vivo tests. The antibodies generated by the 76-amino
acid
fragment showed to confer higher protection than the ones generated by
immunization
with full PBP2a, as previously shown by Ohwada and Senna's works and were
confirmed by the results from the inventors.
The epidemiology of infections caused by MRSA shows the presence of the
predominant clonal types, responsible for outbreaks and epidemics in hospitals
in the
whole world. These clones present a higher colonization and virulence capacity
when
compared to non-epidemic MRSA strains (30).
As a way to validate the obtained results, it was intended to work with
different
MRSA clones (epidemic clones), representatives from most infections
(especially the
hospital ones) caused by MRSA.
CEB strain (Brazilian epidemic clone) is responsible for most infections
caused by
MRSA in Brazilian hospitals (31), being also identified in other countries in
South
America (32, 33), Portugal, and Czechoslovakia (34). The European epidemic
clone
(Iberian-MRSA) is found in European countries and in the United States (35).
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Due to the increase in cases of community infections caused by MRSA, one of
these
strains (WB79 CA-MRSA), isolated in Brazil, was included in the current
trials. These
trials present features differentiated from the ones found in hospitals, with
higher
virulence (presence of Panton-Valentine leukocidin) and profile of resistance
to
antimicrobials different from the one of hospital MRSA strains (36). Recently,
an
outbreak of infections by CA-MRSA in a population of men practicing sex with
other
men was identified in the United States (37). From this date, we can say that
infections
caused by MRSA assume a STD (sexually transmitted disease) character.
The protection results conferred by administration of anti-PBP2a antibody
showed
efficacy against all the different tested clones, which led us to believe in
their
applicability for infections (community or hospital) caused by any MRSA type.
Some significant features of the product of the current invention need to be
commented.
(i) In vitro protection results cause to believe that the involved mechanism
of action -
block of a region close to the molecule active core - is sufficient to inhibit
the bacterial
growth and multiplication. Similarly to the one of beta-lactam antibiotics,
this
mechanism is time-dependent, needing that the bacterium is in the
multiplication phase
to expose the target to the drug action.
(ii) This feature is favored by the pharmacokinetic properties of antibodies,
which
present long plasma half-life (38). Comparing to the antibiotic therapy, this
means an
administration in lower number of doses, presenting treatment facility for the
patient
and maybe lower final cost as consequences. These ideas were confirmed in
practice in
the protection comparative assay with vancomycin (see below).
(iii) The inhibition of in vitro bacterial growth also means that the antibody
does not
need the traditional ancillary antigen-antibody response mechanisms, such as
opsonization and complement activation. Considering that most hospital
infections
occur in patients that has immunodepression status, this product feature is
extremely
significant.
(iv) It is known that Staphylococcus aureus presents a protein A on its
surface. This
molecule characteristically binds itself to the Fc region of immunoglobulins,
impeding
the opsonizing activity of the immune system (38). Due to the results obtained
in our
trials, we believe that the administered antibody concentration was able to
saturate the
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protein A existing at the bacterial surface, not preventing the PBP2a block by
the
administered antibodies.
Systemic infections caused by MRSA have been treated with use of
glycopeptides,
especially vancomycin. However, this drug presents a range of side effects,
with
immunotoxicity (42), ototoxicity, nephrotoxicity, and transient neutropenia
(43) among
them, and need administration for long periods, presenting high final
treatment cost.
By using a murine infection model, it was possible to compare a treatment with
vancomycin versus monoclonal antibody and the combination of the two drugs.
The
obtained results indicated that an antibody dose had action similar to or
higher than 5
vancomycin doses and that the simultaneous administration of the two drugs was
very
more efficient in eliminating the bacterium than the isolated administration.
These data
are extremely significant, as it is possible to think about a lower treatment
cost with the
antibody and a more efficient recovery of patients with severe status with the
administration of vancomycin and antibody.
In addition to the use of anti-PBP2a monoclonal antibodies for treating
infections by
MRSA, the current invention still considers the following applications:
(i) the results of recognition of a protein with molecular weight close to the
one of
PBP5 in Enterococcus sp. strain by anti-PBP2a monoclonal antibody indicate
that it is
possible to obtain a protective response against this pathogen with the
antibody
administration. PBP5 presents homology with PBP2a, with both being classified
as
class B multimodular PBPs, with low affinity for beta-lactams (40).
Enterococci are
bacteria that cause severe hospital infections, presenting high degree of
intrinsic and
acquired resistance to antimicrobials. Strains resistant to vancomycin (VRE)
represent a
reservoir of genes of resistance to glycopeptides and can be transferred to
other
pathogens (41).
(ii) these antibodies can have applicability for PBP2a identification by
immunodiagnostic tests. For example, the use of immune tests based on
agglutination of
latex particles bound to the antibody can provide a result that foresees
resistance to all
beta-lactam antibiotics in few hours. In the conventional tests of sensitivity
to
antimicrobials (such as antibiotic sensitivity testing), these results are
only released after
12 to 24 hours.
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(iii) once monoclonal antibodies have been successfully used in topic
applications
against chronic sores (infliximab, see reference Streit et al., and WO
1999041285 Al),
these antibodies might be topically administered to promote nasal
decolonization by
MRSA and for treatment of skin lesions caused by this pathogen.
Based on the knowledge and issues found in the state of art, the current
inventors
performed immunizations in animals with a DNA vaccine with constructions
corresponding to the gene mecA (SEQ ID NO.: 2) without the membrane fixation
region and immunizations with a 76-amino acid internal region of the
transpeptidase
domain (SED ID NO.: 3) comprising the enzyme active core.
The immunizations were performed in the same conditions presented in the works
of
Ohwada et al. and Senna et al., who developed a DNA vaccine against PBP2a,
using the
full sequence (except the membrane fixation region) of the gene mecA and an
internal
fragment of the transpeptidase domain. The immunizations were performed with 4
doses, and the immunized animals and a non-immunized control group were
challenged
by a systemic infection with MRSA and a determination of the number of
bacteria
present in the kidneys of animals in two distinctive periods. The obtained
results
showed that the immunization with the transpeptidase fragment conferred a
greater
reduction in the number of bacteria present in the animal kidneys than the one
in the
animals immunized with gene mecA.
Thus, we see that the antibodies generated against the transpeptidase fragment
provided a better protection in the used conditions (DNA vaccine in murine
model) than
the one by antibodies against the complete sequence of PBP2a. The
transpeptidase fragment
includes the enzyme active core STQK (SEQ ID NO.: 4).
Consequently, based on the found results, the inventors directed the invention
for
producing monoclonal antibodies able to recognize and bind to the PBP2a
protein and
other proteins presenting sequences homologous to PBP2a protein and using the
transpeptidase fragment that includes the enzyme active core.
The invention will be now described with reference to examples, which must not
be
considered as limiting.
Example 1
MATERIALS AND METHODS:
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1. Bacteria:
The following methicillin-resistant Staphylococcus aureus strains were used:
Iberian-
MRSA, COL-MRSA (ceded by Unite des Agents Antimicrobiens - Institut Pasteur
[Unit of Antimicrobial Agents - Pasteur Institute], Dr. Patrice Courvalin),
WB79 CA-
MRSA, and CEB-MRSA (ceded by Dr. Agnes Figueiredo, Instituto de Microbiologia
[Microbiology Institute] of UFRJ); a vancomycin-resistant Enterococcus
faecalis strain;
and a methicillin-sensitive Staphylococcus aureus (MSSA) strain. Escherichia
coli
strains BL21 DE3 (Novagen) and TOP10 (Invitrogen) were also used as control.
2. Animals:
Female Balb/C mice, 4 to 8 weeks old, obtained from CECAL-FIOCRUZ and housed
at LAEAN-BioManguinhos were used in the immunization and in vivo protection
assays.
3. Immunization:
The mice (4 animals) received an initial dose of 100 micrograms of pCI-Neo
plasmid:
fragment of MRSA mecA gene (18), followed by a 10-microgram dose of purified
recombining protein 14 days later, corresponding to an internal region of the
MRSA
PBP2a (21), emulsified in complete Freund adjuvant, followed by other dose 14
days
later, emulsified in incomplete Freund adjuvant. Fourteen days later, the
animal with
best immune response (evaluated by enzyme immunoassay - ELISA) received an
intravenous dose of 10 micrograms of purified protein diluted in PBS
(phosphate
buffered saline). Three days after the IV injection, the animal was subjected
to
euthanasia by asphyxia in CO2 (CEUA L0009-07 Protocol - FIOCRUZ), and the
spleen was aseptically removed for cell fusion. The serum of the animal with
best
immune response was used as positive control for other immunological tests.
4. Production of monoclonal antibodies:
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Lymphocytes removed from spleen were subjected to fusion with SP2/0-Ag14
myeloma
cells (ATCC 1581), using polyethylene glycol for fusion, and were cultured in
hypoxanthine-aminopterin-thymidine medium at 37 C in atmosphere of 10% CO2,
according to the protocol for producing monoclonal antibodies in Current
Protocols in
Immunology (22). The resulting hybridomas were evaluated by ELISA test after
14 days,
using the purified recombining protein as antigen, according to what is
described below.
The best hybridomas were subjected to cloning, with selection of the best
clones by ELISA
and these latter being stored in liquid nitrogen.
5. Enzyme immunoassay - ELISA:
Maxisorb 98-well plastic plates were sensitized with 500 nanograms/well of
recombining protein (PBP2a fragment) in carbonate/bicarbonate buffer and
incubated at
4 C for the whole night. In the following day, the plates were washed three
times with
PBS containing 0.05% of TweenTm 20 and blocked in PBS and 5% skimmed milk for
two hours at 37 C. The samples to be analyzed (serum of immunized animals
diluted at
1:100 or cell culture supernatants) and incubated for 2 hours at 37 C were
used. The
plate was washed three times with PBS and Tween 20 (0.05%) again, and the anti-
Ig
conjugate (anti-mouse BRP Ig SIGMA A 0412) was added at 1:5000 dilution,
followed
by incubation at 37 C for 90 minutes. After this period, the plate was washed
three times
with PBS and Tween 20 (0.05%), with addition of TMB peroxidase color developer
(BioRad) and incubation for 15 minutes protected from light. The reaction was
interrupted by addition of 0.5 N H2504, and the reading was performed at 450
nm. A
1:200 diluted hyperimmune polyclonal serum was used as positive control.
5.1. Avidity assays based on enzyme immunoassay
5.1.1. Avidity assay with urea (Niederhouser et al. - 5.1):
The protocol is similar to the immunoassay one (5), with the following
modifications:
after the sample incubation (100 ng of clone 10 purified monoclonal antibody
and 2.0 ng
of clone 38 purified monoclonal antibody) for two hours at 37 C, the samples
were
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subjected to three washings with 8 M urea in PBS and Tween 20 (0.05%),
followed by
four washings in PBS and Tween 20 (0.05%), presenting the same normally
processed
sample (without treatment with urea) as control. After the reading of sample
optical
densities, the avidity rate was calculated by the ratio of the reading with
urea divided by
the reading without urea, multiplied by 100 (percentile result).
5.1.2. Avidity assay with ammonium thiocyanate (Goldblat et aL - 5.2)
The protocol is similar to the immunoassay one (5), with the following
modifications:
after the sample incubation (100 ng of clone 10 purified monoclonal antibody
and 2.0
ng of clone 38 purified monoclonal antibody) for two hours at 37 C, the
samples were
subjected to treatment with ammonium thiocyanate for 30 minutes at 37 C in the
following concentrations: 3 M; 1.5 M; 1.0 M; 0.75 M; 0.50 M; 0.25 M; and 0.125
M. A
sample non-treated with ammonium thiocyanate was used as control for each
clone.
After the reading of sample optical densities, the avidity rate was calculated
by the
following formula: AR (avidity rate) = [(log 50 - log A) x (B - A)/log B - log
A] + A;
where log 50 = 1.70. A is the lowest ammonium thiocyanate concentration that
results
in an absorbance reduction lower than 50%, and B is the highest ammonium
thiocyanate
concentration that results in an absorbance reduction higher than 50%.
6. Monoclonal antibody production and purification:
A sample of previously selected clones 10 and 38 was grown in a serumless
medium
(GIBCO VP-SFM) with addition of 1% BSA in 100-mL vials in a stove with
atmosphere of 10% CO2. The supernatants were subjected to centrifugation,
followed
by filtration in 0.22-micrometer filters and purified in high performance
chromatography (HPLC) with SelectSure protein A MAB resin (GE). The antibodies
were neutralized at pH 7.0 with 1 M Tris, pH 10.0, dialyzed against PBS 0.5x
in
deionized water. The samples were subjected to lyophilization process,
resuspended in
deionized water, quantified by the Lowry method, and evaluated by
electrophoresis in
polyacrylamide gel.
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7. Target recognition:
7.1. In vitro PBP2a recognition - immunoblotting:
A MRSA (CEB) strain, a methicillin-sensitive Staphylococcus aureus (MSSA)
strain,
a vancomycin-resistant Enterococcus faecium strain, and the BL-21 DE3
Escherichia
coil strain were grown in exponential phase. One mL of each sample was
centrifuged
and lysed by agitation in glass beads in a mini-Bead Beater device (Biospect
Products),
3 times for 30 seconds. One aliquot of each sample was subjected to
electrophoresis in
12% denaturing polyacrylamide gel (SDS-PAGE), and the proteins were later
transferred to a nylon membrane (Hybond N-BioRad). The membrane was blocked
under mild agitation for two hours in PBS buffer containing 10% skimmed milk
and 1%
BSA (bovine serum albumin). The membrane was washed three times in PBS and
Tween 20 (0.05%) and three times in PBS. Then, the latter was placed in
incubation for
two hours, with supernatant of anti-PBP2a monoclonal antibody diluted in PBS
at 1:1
ratio. After the incubation, the membrane was washed as previously described,
and the
alkaline phosphatase conjugate (murine anti-IgG antibody - Sigma A3688) at
1:15,000
ratio and incubated for ninety minutes. After this period, the washing in PBS
was
performed again, and the development with Western Blue substrate for alkaline
phosphatase (Promega) was performed.
7.2. PBP2a recognition at the bacterium surface - flow cytometry:
A MRSA (CEB) strain was grown in stationary (ON) or exponential phase. The
samples were washed in PBS lx and resuspended at a D0600 of 0.6 (-108
bacteria/mL).
These latter were centrifuged in 1 mL (108 bacteria) and resuspended in 0.5%
BSA and
a 1:10 dilution of normal serum (murine/human). Then, the samples were
incubated for
30 min at 4 C, and later, the concentrates (pellets) were washed 2 times in
PBS and
resuspended in 100 microliters of PBS containing 0.5% BSA and a 1:10,000
dilution of
anti-PBP2a monoclonal antibody and incubated for 30 minutes at 4 C. After this
step,
the samples were washed as previously again and resuspended in 100 microliters
of
PBS and 0.5% BSA and a dilution (1:1000) of PE (phycoerythrin) mouse anti-Ig
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conjugate, later being incubated at dark for 30 min at 4 C. Then, the samples
were
washed again and fixated for 15 minutes at 4 C in PBS containing 2%
paraformaldehyde. After this preparation, the samples were analyzed in a flow
cytometer (Becton and Dickinson - FACScalibur).
8. In vitro protection assays - determination of minimum inhibitory
concentration:
MSRA strains (CEB, Iberian, COL, and CA) were grown in exponential phase up to
600 nm of optical density equal to 0.5. The applied inoculum was adjusted for
containing approximately 100,000 bacteria. Milner Hinton broth, bacterial
inoculum,
and growing amounts of purified anti-PBP2a monoclonal antibody were added to
test
tubes or 24-cavity plates. The plates or tubes were subjected to incubation at
37 C for
12 hours. After this period, presence or absence of turbidity was observed in
the
samples. The minimum inhibitory concentration was considered as the lower
antibody
amount able to inhibit the bacterial inoculum growth (100,000 bacteria).
9. In vivo protection assays:
9.1. Determination of lethal dose and LD50 for MRSA strains.
The determination of lethal dose and LD50 were performed according to the Reed-
Muench method (23) for MRSA strains (CEB, Iberian, WB79 CA, and COL). Groups
of 8-week female Balb/C mice were inoculated by intraperitoneal route with
growing
bacteria doses and observed for 7 days. The animals that survived after this
period were
subjected to euthanasia, according to the established animal welfare rules.
9.2. Systemic infection and bacterial renal quantification assays.
MRSA strains (CEB, Iberian, and WB79 CA) were grown in exponential phase
(D0600 ¨0.6), washed and resuspended in sterile PBS lx at D0600 ¨0.5,
corresponding
to approximately 2 x 108 bacteria. This concentration was calculated by
dilution and
seeding on BHI agar plates containing 10 micrograms of oxacillin/mL. Female 8-
week
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CA 02770771 2012-02-10
Balb/C mice received one intraperitoneal dose of 400 micrograms of purified
anti-
PBP2a monoclonal antibody in the first day. In the sixth day, the animals were
subjected to euthanasia and the kidneys were aseptically removed. Then, the
kidneys
were homogenized in 1 mL of sterile Luria broth and successively diluted in
series of
10. One hundred microliters of each dilution were seeded on BHI agar plates
containing
micrograms/mL of oxacillin and incubated for 24 hours at 37 C, with a count of
resulting colonies and calculations of total dilutions being performed.
9.3. Survival assay.
MRSA strains were grown in the same previously described conditions, but an
inoculum adjustment was performed for the previously established LD50. Female
8-
week Balb/C mice received one intraperitoneal dose of 500 micrograms of
purified anti-
PBP2a monoclonal antibody in the first day. In the following day, these
animals plus a
control group were infected with one dose of about 2.5 to 6.0 x 108 bacteria
by
intraperitoneal route, according to the LD50 of each strain. The animals were
observed
for 10 days, with the survivors being subjected to euthanasia.
10. Comparative in vivo protection study of anti-PBP2a monoclonal antibody and
vancomycin - renal quantification:
Assay I
Four groups of female 8-week Balb/C mice (4 animals per group) received an
infective dose of 6.0 x 107 bacteria (CEB-MRSA) by intraperitoneal route. The
animals
received doses of purified monoclonal antibody (MAB), vancomycin, MAB +
vancomycin, and negative control, according to the groups below:
group 1: MAB (400 micrograms) (first day)
group 2: vancomycin (150 micrograms, intramuscular route, 12/12 hours)
group 3: vancomycin + MAB (350 micrograms) (1 day after infection)
group 4: control
CA 02770771 2012-02-10
The first doses of antibody and vancomycin were administered 4 hours after the
administration of the infective dose. The animals were subjected to euthanasia
in the
fourth day, and the kidneys were aseptically removed and subjected to renal
qualification, according to what was previously described.
Assay II
This assay was performed in the same way as the previous one, but, with a
lower
infective dose (7.0 x 106 bacteria), with the group 1 being treated with 500
micrograms
of purified monoclonal antibody; group 2 being treated with vancomycin (150
micrograms, intramuscular route, 12/12 hours; 5 doses); group 3 being treated
with
vancomycin + 500 micrograms of monoclonal antibody; and group 4 being control
(non-treated animals).
11. Identification complementarity-determining regions (CDRs) of light and
heavy
chains of the anti-PBP2a monoclonal antibody
11.1. mRNA extraction from hybridoma cells
A 10-mL centrifugate (pellet) of a cell culture of monoclonal antibody-
producing
hybridoma was processed for mRNA extraction using the RNeasy Minikit kit
(Qiagen).
11.2. cDNA obtainment
Reverse transcriptase reaction: M-MLV reverse transcriptase kit (Invitrogen)
was
used for obtaining complementary DNA, following the manufacturing guidelines.
11.3. Amplification of VH and VL chains by polymerase chain reaction (PCR)
The reactions were performed using the starter sequences (primers) below.
SEQ Sequence Use
16
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ID NO.
FOR HEAVY CHAIN
18 5' ATG(GA) VH
A(GC)TT(GC)(TG)GG(TC)T(AC)A(AG)CT(GT)G(GA)TT Amplification
3'
19 5 VH
ATG(GA)AATG(GC)A(GC)CTGGT(CT)(TA)T(TC)CTCT Amplification
3'
20 5' GATGTGAAGCTTCAGGAGTC 3' VH
Amplification
21 5' CAGGTGCAGCTGAAGGAGTC 3' VH
Amplification
22 5' CAGGTGCAGCTGAAGCAGTC 3' VH
Amplification
23 5' CAGGTTACTCTGAAAGAGTC 3' VH
Amplification
24 5' GAGGTCCAGCTGCAACAATCT 3' VH
Amplification
25 5' GAGGTCCAGCTGCAGCAGTC 3' VH
Amplification
26 5' CAGGTCCAACTGCAGCAGCCT 3' VH
Amplification
27 5' GAGGTGAAGCTGGTGGAGTC 3' VH
Amplification
28 5' GATGTGAACTTGGAAGTGTC 3' VH
Amplification
29 5' TGGACAGGGATCCAGAGTTCCAGGTCACT 3' VH
(gamma Amplification
1)
FOR LIGHT CHAIN
30 5' GACATTGTGATGACCCAGTCT 3' VL
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Amplification
31 5' GATGTTTTGATGACCCAAACT 3' VL
Amplification
32 5' GATATTGTGATAACCCAG 3' VL
Amplification
33 5' GACATTGTGCTGACCCAATCT 3' VL
Amplification
34 5' GATATTGTGCTAACTCAGTCT 3' VL
Amplification
35 5' GATATCCAGATGACACAGACT 3' VL
Amplification
36 5' GACATCCAGCTGACTCAGTCT 3' VL
Amplification
37 5' CAAATTGTTCTCACCCAGTCT 3' VL
Amplification
38 5 'CAGGCTGTTGTGACTCAGGAA 3' VL
Amplification
39 5' TACAGTTGGTGCAGCATC 3' VL
(kappa Amplification
18)
11.4. Sequencing of light and heavy chains of anti-PBP2a monoclonal antibody
Sequencing Steps:
I. Amplification
This was performed using the previous starter sequences, defined by SEW ID
NO.:
18 to SEQ ID NO.: 39.
II. Sequencing
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CA 02770771 2016-12-20
The device ABI Prism 3100, Genetic Analyser (Hitachi) was used.
11.5. Sequence analysis and identification of light and heavy chain CDR
The obtained DNA sequences were analyzed with the aid of DNA Star program, and
were translated to amino acid sequences (ExPASy site - Translate program) for
later
analysis by ICabafs (24) and Chotia's (25) algorithms for identification of
light and heavy
chain CDRs.
12. Determination of association and dissociation constants for monoclonal
antibodies (clones 10 and 38 by surface plasmon resonance [SPR] [BiacoreTMl
method)
SPR measurements were performed using a CM-5 sensor in a Biacore X (Biacore
AB,
Uppsala, Sweden) device. Binding reagents and HBS-EP buffers (10 mM hepes, 150
mM
NaC1, 3 mM EDTA, 0.005% P20 [Tween 0, pH 7.4]) were acquired with the company
GE
Healthcare.
Example 2
1. Obtainment of murine anti-PBP2a monoclonal antibodies:
A group of animals was subjected to the immunization protocol according to
what was
previously described. The result evaluated by ELISA test is described in
Figure 1.
After the fusion process (fusion of number 90-LATAM), the supernatant from 96
cavities (hybridomas) was analyzed by ELISA test. From this total, the five
best samples
were selected and the cells were expanded (cloning). Then, again, the
resulting
supernatants were analyzed by ELISA. Positive samples were validated by
immunoblotting against the purified recombinant protein (PBP2a) for validating
the results
obtained by ELISA. The final result is in Table I.
Figure 1 reveals the result from the immunoenzyme test (ELISA) of the sera of
the
immunized animals for producing anti-PBP2a antibodies. Each dashing
corresponds to
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the 1:100 diluted serum of the immunized animals. Positive control serum is in
angular
dashing [ n-11-]. First bar of each dashing: pre-immune serum; second bar:
serum after
fourth immunization; and third bar: serum after fifth immunization.
Table I. Clonings of fusion 90: final balance
Cloned Total number Positive Positive clones Selected
hybridomas of clones clones (immunoblotting) clones
(ELISA)
77 70 2 1 (clone 38) 1
68 108 10 1 (clone 10) 1
79 91 Zero - -
17 101 Zero - -
70 48 2 Zero -
418 14 2 2
From the selected clones, clone 77-38 was subjected to recloning process for
verifying the cell stability for secreting monoclonal antibodies. From the 50
analyzed
cavities, all presented positive result by ELISA test. These clone were
expanded and are
stocked in liquid nitrogen at LATAM (Laboratory of monoclonal antibody
technology)
facilities.
2. Growth, production, and purification of monoclonal antibodies:
The process was standardized according to what was previously described. The
obtained output is approximately 4 milligrams of monoclonal antibody for every
100
mL of supernatant subjected to the purification process. The obtained results
can be
seen below.
In Figure 2, we can see a polyacrylamide gel with raw samples and after
purification.
This Figure 2 shows the non-denaturing polyacrylamide gel with supernatant
samples
before (column 1) and after purification (2) in HPLC column with MAb
SelectSure
CA 02770771 2012-02-10
resin. Columns 3 to 9 correspond to obtained purified sample fractions. The
arrow
indicates the approximate size of 150 kDa.
3. Functional characterization of monoclonal antibodies:
3.1. In vitro PBP2a recognition - immunoblotting
In order to investigate the capacity for antibody recognition by the target in
the
pathogen (PBP2a and similar sequences), the immunoblotting test was performed
with
bacteria presenting PBP2a (CEB and COL MRSA), a MSSA (methicillin-sensitive
Staphylococcus aureus) strain (not presenting PBP2a), a vancomycin-resistant
Enterococcus sp. strain (presenting PBP5), a transpeptidase with low-affinity
for beta-
lactam antibiotics (homologous to PBP2a), and an Escherichia coli strain
(where the
recombinant protein was generated) as negative control. The obtained results
show that
the antibody was able to recognize a protein with molecular weight of
approximately 76
kDa in MRSA and Enterococcus sp. strains, corresponding to the size of PBP2a
and
PBP5, respectively. No reactivity of the monoclonal antibody was seen with
proteins of
methicillin-sensitive Staphylococcus aureus (PBP2a-negative) neither with
Escherichia
coli strain. The results can be seen in Figure 3 (immunoblotting against MRSA
and
MSSA).
The result of the immunoblotting test of MRSA and MSSA lysates against the
supernatant containing anti-PBP2a monoclonal antibodies is shown in Figure 3.
1. - molecular weight marker (Kaleidoscope);
2. - MSSA sample grown in exponential phase;
3 and 4. - MRSA sample grown in exponential phase;
5. - MSSA sample grown in stationary phase (overnight); and
6 and 7. - MRSA samples grown in stationary phase. The left arrow indicates
PBP2a
molecular weight.
3.2. PBP2a recognition on the bacterium surface - flow cytometry
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The objective of the flow cytometry test is to validate the capacity for
target
recognition on the bacterium in its native form by the monoclonal antibody. In
previous
tests (immunoblotting), we observed the target recognition on proteins
subjected to a
denaturation process, which occurs during the protein separation by
electrophoresis in
denaturing polyacrylamide gel. Again, we analyze a negative control strain
(MSSA,
PBP2a-negative) and a MRSA strain (CEB) grown in exponential and stationary
phases.
The obtained results show that the antibody is able to recognize the target on
the
bacterial surface in both conditions. The presence of protein A on
Staphylococcus
surface was not able to inhibit the antibody binding with PBP2a. The obtained
results
can be seen in Figure 4.
Figure 4 shows the results of the flow cytometry of MRSA (CEB) and MSSA
bacteria incubated with anti-PBP2a monoclonal antibody and marked with
phycoerythrin (PE). In (1), MSSA bacteria; in (2), MRSA grown in stationary
phase; in
(3), MRSA grown in exponential phase. MRSA populations present a right shift,
corresponding to an increase in marked cells by the fluorescent conjugate.
4. Evaluation of protection conferred by anti-PBP2a monoclonal antibody:
4.1. In vitro protection tests
Determination of minimum inhibitory concentration (MIC)
These assays aim to evaluate the antibody capacity for directly binding itself
to the
target in a closed system. For monoclonal antibodies with therapeutic
finality, positive
results have extreme significance, as they mean that the antibody is able to
recognize
the target and to block the bacterial growth without the aid of the host
immune system,
such as complement activation and opsonization mechanisms, resulting from the
combined action with the innate and adaptative immune system of the host. CEB,
COL,
and Iberian MRSA strains were evaluated, where similar MIC values
(approximately
500 micrograms) were seen in the evaluated conditions. These data indicate
that
regardless of the genetic background of the different MRSA strains, the
antibody doses
needed for blocking the growth are not the same. These results can be seen in
Figure 5.
22
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Figure 5 shows the test of the in vitro protection (MIC - minimum inhibitory
concentration) conferred by anti-PBP2a purified monoclonal antibody and
vancomycin
against an inoculum of 105 cells from different MRSA strains. An absence of
turbidity
indicates that there was no bacterial growth in the analyzed conditions.
1A: CEB MRSA (Brazilian epidemic clone) + 250 ftg of antibody;
2A: CEB MRSA + 500 p,g of antibody;
3A: CEB MRSA + 750 fig of antibody;
4A and 6A: negative controls of CEB MRSA strain.
1B: COL MRSA + 250 fig of antibody;
2B: COL MRSA + 500 fig of antibody;
3B: COL MRSA + 750 fig of antibody;
4B and 6B: negative controls of COL MRSA strain.
1C: Iberian MRSA (European epidemic clone - EEC) + 250 fig of antibody;
2C: EEC MRSA + 500 jig of antibody;
3C: EEC MRSA + 750 fig of antibody;
4C and 6C: negative controls of EEC MRSA strain.
1D: CEB MRSA + 150 fig of vancomycin;
2D: CEB MRSA + 300 fig of vancomycin;
3D: CEB MRSA + 500 fig of vancomycin;
4D: CEB MRSA + 750 fig of vancomycin;
5D and 6D: negative controls.
4.2. In vivo protection tests
4.2.1. Determination of lethal and sublethal doses for CEB, Iberian, WB79 CA,
and COL MRSA strains:
These assays were necessary in order that the in vivo protection was evaluated
in the
two used models - the renal quantification by sublethal dose one and the
survival test
after systemic infection with a larger inoculum of bacteria - able to cause
death in about
50% of the animals (LD50). The chosen route was intraperitoneal, due to the
23
CA 02770771 2012-02-10
administration facility and the absence of losses. An adaptation of Reed-
Muench
method was used for conducting the assay, with two animals per condition
(infective
bacterial dose in growing concentrations) for determining LD50 and sublethal
doses, and
three different doses were tested, once we had previous knowledge on the mean
lethal
doses for Staphylococcus aureus. COL MRSA strain, the first MRSA clone to have
its
genome sequenced, is used as reference strain for studies on this pathogen.
However, it
showed to be little virulent, needing high infective doses in relation to the
other MRSA
clones to cause infection in animals. Therefore, it was not used in protection
assays.
4.2.2. Renal protection assays after systemic infection with sublethal dose
Using a renal quantification model after infection, these assays enable to
evaluate the
antibody capacity to reduce the presence of bacteria in vital organs (kidneys)
after a
systemic infection. A reduction higher than 3 log (1000 times) was reached in
three
independent assays with virulent MRSA strains from different genetic
backgrounds. In
these assays, the animals received a previous dose of 500 micrograms of
antibody. The
protection conferred by a lower antibody dose (250 micrograms) was evaluated
in the
assay with CA-MRSA strain, where a bacterial reduction was also observed, but
lower
than the one obtained with the 500-microgram dose. The results can be seen in
Figures
6A, 6B, and 6C.
Figure 6A shows the results of renal quantification in animals treated and non-
treated
with anti-PBP2a monoclonal antibody and subjected to systemic infection with a
sublethal dose of MRSA CEB strain. In horizontal stripes: log of concentration
of
bacteria isolated from the kidneys of each non-treated animal. In checkered
pattern: log
of quantity of bacteria isolated from the kidneys of each animal treated with
the
antibody. Bacterial quantification: controls: Cl: 2000 bacteria; C2: 29,000
bacteria; C3:
220,000 bacteria; C4: 52,000 bacteria (mean of 75,750 bacteria). Treated
(protected)
animals: Pl: 20 bacteria; P2, P3, and P4: 10 bacteria (mean of 12.5 bacteria).
Reduction
in the quantity of bacteria recovered from treated animals in relation to non-
treated
ones: 6060 times.
Figure 6B shows the results of renal quantification in animals treated and non-
treated
with anti-PBP2a monoclonal antibody and subjected to systemic infection with a
24
CA 02770771 2012-02-10
sublethal dose of Iberian MRSA strain (European epidemic clone). In horizontal
stripes:
log of concentration of bacteria isolated from the kidneys of each non-treated
animal. In
large checkered pattern: log of quantity of bacteria isolated from the kidneys
of each
animal treated with the antibody. Bars in small checkered pattern indicate the
respective
obtained means. Bacterial quantification: controls: Cl: 210,000 bacteria; C2:
44,000
bacteria; C3: 300,000 bacteria; C4: 290,000 bacteria (mean of 211,000
bacteria).
Treated (protected) animals: P1: 80 bacteria; P2: 200 bacteria; P3: 10
bacteria; and P4:
60 bacteria (mean of 87.5 bacteria). Reduction in quantity of bacteria
recovered
from treated animals in relation to the non-treated ones: 2420 times.
Figure 6C shows the results of renal quantification in animals treated and non-
treated
with anti-PBP2a monoclonal antibody and subjected to systemic infection with a
sublethal dose of WB79 CA-MRSA strain (Brazilian community strain). Five first
bars
(in xx, horizontal dashing, and large checkered pattern): log of concentration
of bacteria
isolated from the kidneys of each non-treated animal. First bar (in xx):
estimate in
relation to an animal killed before the euthanasia. Bars 6, 7, 8, and 9 (in
horizontal
dashing and checkered pattern): log of quantity of bacteria isolated from the
kidneys of
animals treated with 250 1..tg of anti-PBP2a monoclonal antibody. Bars 10, 11,
12, and
13 (in triangles): log of quantity of bacteria isolated from the kidneys of
each animal
treated with 500 1,1g of antibody. Checkered bars (5th, 9th,
and 13th bars) indicate the
respective obtained means. Bacterial quantification: controls: Cl: 650,000
bacteria; C2:
26,000 bacteria; C3: 17,000 bacteria; C4: 500,000 bacteria (dead animal
estimate)
(mean of 231,000 bacteria). Animals treated with 250 jig of antibody: P1:
zero; P2:
5,400 bacteria; P3: 830 bacteria; and P4: 10 bacteria (mean of 1,560
bacteria). Animals
treated with 500 jig of antibody: P1: 80; P2: zero; P3: 210; P4: 80 bacteria
(mean of
92.5), Reduction in quantity of bacteria recovered from treated animals in
relation
to the ones non-treated with 250 jig of antibody: 149 times. With 500 2,497
times.
4.2.3. Survival assays
In this assay type, we evaluate the protection conferred by the antibody to
animals
after an infection with a bacterial load able to kill 50% of the animals
(LD50) or more.
CA 02770771 2012-02-10
The protection against the three strains used in the renal quantification
assay was
evaluated. A significant reduction in (i) survival time and (ii) the very
survival of
animals under treatment with anti-MRSA monoclonal antibodies was observed in
the
three independent assays.
In the assay with CEB MRSA strain, 70% of the animals under treatment survived
the infection, against only 10% of the control group (non-treated). In the
assay with
Iberian MRSA strain, the obtained results were similar, with 60% of protection
in
animals under treatment and 100% of death in control group. In the assay with
CA
MRSA strain, a protection of 100% was seen, compared to 70% in control
animals.
These results can be seen in Figures 7a, 7b, and 7c.
Figure 7A shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a dose of 2.3 x 108 bacteria (CEB MRSA)
administered by
intraperitoneal route (LD50).
Figure 7B shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a dose of 4.2 x 108 bacteria (Iberian MRSA)
administered by
intraperitoneal route (¨LD5o).
Figure 7C shows the survival curve of treated (protected) and non-treated
(control)
animals after infection by a dose of 1.1 x 109 bacteria (WB79 CA-MRSA)
administered
by intraperitoneal route (¨LD50).
4.2.4. Comparative study on protection conferred by monoclonal antibody versus
vancomycin
As vancomycin is a first choice antimicrobial for treatment of severe
infections by
MRSA, a comparative study on protection was conducted in a model different
from the
previous ones. In this study, the animals were infected and the administration
of the
antimicrobial or monoclonal antibodies was started just after four hours of
infection.
The study was conducted with three distinct groups: one treated with
vancomycin, other
treated with monoclonal antibodies, and a third one with simultaneous
administration of
antimicrobial + antibodies. Vancomycin doses were adjusted and administered in
a way
similar to the cases of infections in humans (500 mg every 12 hours).
26
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The obtained results indicated that there was a reduction of around 15 times
in the
quantity of bacteria present in the kidneys of animals treated with
antimicrobial or
antibodies three days after the infection. However, a reduction of 4,617 times
was seen
in the group that received treatment with antimicrobial and antibodies.
Based on these results, we can conclude that the protection conferred by an
antibody
dose corresponded to five vancomycin doses and that the simultaneous
administration
of vancomycin and antibodies was very efficient in reducing the bacterial load
observed
in the kidneys of infected animals. This study was repeated with a little
lower infective
dose in order that we could better observe the protective action of anti-MRSA
monoclonal antibodies both isolated and in association with vancomycin (See
figures
8).
After the conduction of the second assay with a lower infective dose, the
obtained
results confirmed the initial results. The protection conferred by the
treatment with
monoclonal antibody caused a reduction of 89 times, which was higher than the
protection obtained with the treatment with 5 vancomycin doses (reduction of
35 times).
However, the most significant reduction result was seen in the group treated
with
antibody + vancomycin, causing a reduction of 450 times.
Figure 8A shows the bacterial quantification in kidneys of animals treated
with anti-
PBP2a monoclonal antibody, vancomycin, and association of antibody +
vancomycin
and in non-treated animals after infection with 6.0 x 107 bacteria (CEB MRSA).
The
treatment beginning happened 4 hours after the infection. Vancomycin was
administered every 12 hours (5 doses). Bars 1, 2, 3, 4, and 5 (dashing): log
of
concentration of bacteria recovered from non-treated animals (controls). Cl:
7,000,000;
C2: 295,000; C3: 380,000; C4: 3,200,000 (mean: 2,718,750 bacteria). Bars 6, 7,
8, 9,
and 10 (checkered pattern): log of concentration of bacteria recovered from
animals
treated with 400 jig of anti-PBP2a monoclonal antibody. P1: 4,200; P2:
310,000; P3:
330,000; P4: 90,000 (mean of 183,550 bacteria). Bars 11, 12, 13, 14, and 15
(spheres):
animals treated with vancomycin. P1: 110,000; P2: 58,000; P3: 500,000; P4:
21,000
(mean of 172,250 bacteria). Bars 16, 17, 18, 19, and 290 (triangles): log of
concentration of bacteria recovered from animals treated with antibody (300
1.1.g) +
vancomycin. Pl: 1,100; P2: 700; P3: 450; P4: 90 (mean of 585 bacteria).
27
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Figure 8B shows the bacterial quantification in kidneys of animals treated
with anti-
PBP2a monoclonal antibody (bars 7 to 12), vancomycin (bars 13 to 18), antibody
+
vancomycin association (19 to 24) and of non-treated animals (bars 1 to 6)
after
infection with 7.0 x 106 bacteria (CEB MRSA). Treatment beginning in 4 hours
after
infection. Vancomycin administered every 12 hours (5 doses). Bars 1 to 6: log
of
concentration of bacteria recovered from non-treated animals (controls). Cl:
6,000; C2:
1,000; C3: 500; C4: 118,000; and C5: 1,000 (mean: 25,220 bacteria). Bars 7 to
12: log
of concentration of bacteria recovered from animals treated with 500 lig of
anti-PBP2a
monoclonal antibody. MB1: 450; MB2: 200; MB3: 100; MB4: 20; MB5: zero (mean of
284 bacteria). Bars 13 to 18: animals treated with vancomycin. VC1: 100; VC2:
700;
VC3: zero; VC4: zero; VCS: 2800 (mean of 720 bacteria). Bars 19 to 24: log of
concentration of bacteria recovered from animals treated with antibody (500
+
vancomycin. MBV1: 130; MBV2: 20; MBV3: 10; MBV4: 80; MBV5: 20 (mean of 56
bacteria).
5. Avidity assays
Results of the avidity tests of monoclonal antibodies clones 10 and 38:
Urea protocol avidity:
clone 10: 1.03/1.46 (reading DOs with/without treatment) = 70.5%
clone 38: 1.00/1.21 = 82.6%
Ammonium thiocyanate protocol avidity:
Clone 10 (D0s):
Control: 1.12
Thiocyanate-treated samples:
2 M = 0.046; 1.5 M = 0,047; 1 M = 0.107; 0.75 M = 0.483; 0.5 M = 0.602; 0.375
M =
0.684
Avidity rate: 2.47
Clone 38 (D0s):
Control: 1.22
28
CA 02770771 2012-02-10
Thiocyanate-treated samples:
2 M = 0.056; 1.5 M = 0.062; 1 M = 0.129; 0.75 M = 0.648; 0.5 M = 0.758; 0.375
M =
0.793
Avidity rate: 4.40
It is seen that the avidity rates of clone 38 were higher than the ones of
clone 10 in
both assays. Furthermore, it is verified that, according to both protocols, it
was
necessary to use 50 times more antibody from clone 10 than from clone 38 in
order to
reach a DO close to 1Ø
6. Determination of association and dissociation constants of the monoclonal
antibodies (clones 10 and 38 by surface plasmon resonance method ISPR1
[BIAcore])
The results obtained by SPR method confirmed the preliminary results of
antibody
avidity, where clone 38 showed results higher than the ones of clone 10 again.
According to the data seen in Table II, we see that clone 38 shows affinity
450 times
higher than the clone 10 one.
This affinity is mainly due to its higher association rate, which is about 100
times
higher than the clone 10 one. Due to the very high affinity of clone 38, its
measures
present parameters close to the equipment detection threshold. Yet, due to the
care
in trial planning and conduction, we obtained an excellent adjustment for
trial data
using Langmuir's model, which indicates that the obtained data are reliable.
Figure 9 shows the interaction between recombinant PBP2a (antigen) and
monoclonal antibodies clone 38 (Figure 9A) and clone 10 (Figure 9B). Smoky
dashing
curves represent SPR data in the concentrations according to the right key.
All samples
were analyzed in duplicate, and the 1:1 Langmuir's theoretical model for each
curve is
shown in black under each curve. Response units are represented in the
vertical axis,
and time is represented in the horizontal line in seconds. The closest lines
referring to
the horizontal axis represent the baseline for each sample (negative control).
Table II
29
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Dissociation and interaction constants between antigen (PBP2a) and monoclonal
antibodies clones 10 and 38.
ka (M-1s-11) kd (S-1) KD (nM)
Clone 10 4.42 x 103 5.7 1.16 x 10-4 5.63 x 26.2
1 0-7
Clone 38 5.45 x 105 384 3.13 x i0 2.11 x 5.7 x 10-2
10-10
7. Identification of complementary-determining regions (CDRs) of light and
heavy
chains of anti-PBP2a monoclonal antibody
After the mRNA extraction process in the hybridoma cells of the clone
producing the
used antibodies (clone 38), cDNA obtainment was performed and PCR reactions
were
performed with this material for different light and heavy chain alleles. The
obtained
materials were subjected to sequencing using the same starter sequences
(defined by
SEQ ID NO.: 18 to SEQ ID NO.: 39) used in PCR reactions. Light chain 391 and
heavy
chain 310 were identified in three distinct sequencings. Applying the Kabat's
and
Chotia's algorithms, we obtain the identification of light and heavy chain
CDRs, which
are the target of the attached claims.
We present the sequences of light and heavy chain CDRs below.
SEQ ID NO.: 6 - CDR 1 light chain amino acids.
RSSQSIGHSNGNTYLE
SEQ ID NO.: 7 - CDR 2 light chain amino acids.
KVSNRFS
SEQ ID NO.: 8 - CDR 3 light chain amino acids.
FQGSYVPLT
SEQ ID NO.: 9- CDR 1 light chain DNA.
cgcagcagccagagcattggccatagcaacggcaacacctatctggaa
SEQ ID NO.: 10 - CDR 2 light chain DNA.
CA 02770771 2012-02-10
aaagtgagcaaccgctttagc
SEQ ID NO.: 11 - CDR 3 light chain DNA.
tttcagggcagctatgtgccgctgacc
SEQ ID NO.: 12 - CDR 1 heavy chain amino acids.
GFSITSSSSCVVH
SEQ ID NO.: 13 - CDR 2 heavy chain amino acids.
RICYEGSISYSPSLKS
SEQ ID NO.: 14 - CDR 3 heavy chain amino acids.
ENHDWFFDV
SEQ ID NO.: 15 - CDR 1 heavy chain DNA.
ggattagcattaccagcagcagcagctgctggcat
SEQ ID NO.: 16 - CDR 2 heavy chain DNA.
cgcatttgetatgaaggcagcattagetatagcccgagcctgaaaagc
SEQ ID NO.: 17 - CDR 3 heavy chain DNA.
gaaaaccatgattggifitttgatgtg
Complementary data
Further other assays were conducted, proceeding the invention development.
These
are reported below, through examples.
Example 3
A second study using CEB MRSA strain was conducted, following the same
protocol
described in Example 1, item 7.2, with adding of two pulses of agitation by
vortex, of
15 seconds each one, after each washing; aiming to disaggregate Staphylococcus
aureus
agglomerates and to increase the quantity of PBP2a exposed to antibodies.
The markers FITC (fluorescein isothiocyanate) and PE (phycoerythrin) were
tested,
with control samples (i. pure bacterium, without contact with monoclonal
antibody; and
ii. pure bacterium plus FITC or PE marker) and the sample treated with
monoclonal
antibody and subjected to marking with PE or FITC being analyzed. The readings
in
FACsalibur device were performed in linear mode.
31
CA 02770771 2012-02-10
Figure 10 is a graph of analysis by flow cytometry of MRSA samples in presence
of
FITC-marked anti-PBP2a antibody. Curve (x) corresponds to non-marked sample,
and
curve (y) corresponds to marked sample.
The obtained results showed that about 22% of marked population was detected
by
the device, confirming the recognition of the target (PBP2a) present on the
bacterial
surface by anti-PBP2a antibodies (Figure 10).
Example 4
The inventors still surveyed the protection conferred by anti-PBP2a monoclonal
antibody from methicillin-resistant Staphylococcus aureus against
enterococcus.
According to what was already previously mentioned here, the antibody
recognizes
proteins present in Enterococcus sp. strains, probably PBP5 - a transpeptidase
with low
affinity for beta-lactams, present in all enterococcus strains, with molecular
weight of
approximately 76 kDa (237 amino acids). This enzyme presents homology
referring to
PBP2a of MRSA, according to alignment (ClustalW) related below:
PBP5Efas
MERSNRNKKSSKNPLILGVSALVLIAAAVGGYYAYSQWQAKQELAEAKKTATTFL
NVLSK 60
PBP5efam ---- KHGKNRTGAYIAG--
AVILIAAAGGGYFYYQHYQETQAVEAGEKTVEQFVQALNK 53
PBP2a
MKKIKIVPLILIVVVVGFGIYFYASKDKEINNTIDAIEDKNFKQVYKD 48
* =
PBP5Efas
QEFDKLPSVVQEASLKKNGYDTKSVVEKYQAIYSGIQAEGVKASDVQVKKAKDN
QYTFTY 120
PBP5efam
GDYNKAAEMTSKKAANKSALSEKEILDKYQNIYGAADVKGLQISNLKVDKKDDST
YSFSY 113
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CA 02770771 2012-02-10
PBP2a SSY -----
ISKSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKKVSKNKKRVDA 97
. . . ..... .
PBP5Efas
KLSMSTPLGEMKDLSYQS SIAKKGDTYQIAWKPSLIFPDMSGNDKISIQVDNAKRG
EIVD 180
PBP5efam
KAKMNTSLGELKDLSYKGTLDRNDGQTTINWQPNLVFPEMEGNDKVSLTTQEAA
RGNIID 173
PBP2a QYKIKTNYGNIDRN-
VQFNFVKEDGMWKLDWDHSVIIPGMQKDQSIHIENLKSERGKILD 156
. . . . . . .
PBP5Efas
RNGSGLAINKVFDEVGVVPGKLGSGAEKTANIKAFSDKFGVSVDEIN---
QKLSQGWVQA 237
PBP5efam
RNGEPLATTGKLKQLGVVPSKLGDGDEKTANIKAIASSFDLTEDAIN---
QAISQSWVQP 230
PBP2a RNNVELANTGTHMRLGIVPKNVS
KKDYKAIAKELSISEDYINNKWIKIGYKMIPS 211
** ** =*=** .. = **.. . * ** . .
. .
PBP5Efas DSFVPITVASEPVTELPTG--AATKDTESRYYPLGEACAINR-
VYGTITAEDIEKN¨PE 292
PBP5efam DYFVPLKIIDGATPELPAG--
ATIQEVDGRYYPLGEAAAQLIGYVGDITAEDIDKN¨PE 286
PBP2a
FHFKTVKKMDEYLSDFAKKFHLTTNETESRNYPLEKATSHLLGYVGPINSEELKQK
EYKG 271
* = .. = . * *** =* . * * =*==
33
CA 02770771 2012-02-10
PBP5Efas LS STGVIGKTGLERAFDKELRGQDGGSLVILDDK-
ENVKKALQTKEKKDGQTIKLTIDSG 351
PBP5efam LSSNGKIGRSGLEMAFDKDLRGTTGGKLSITDAD-
GVEKKVLIEHEVQNGKDIKLTIDAK 345
PBP2a
YKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNSNTIAHTLIEKKKKDGKDIQL
TIDAK 331
**. *** .** *= * . * * = * ..*. *=****=
. . . . . . . .
PBP5Efas
VQQQAFAIFDKRPGSAVITDPQKGDLLATVSSPSYDPNKMANGISQKEYDAYNNN
ICDLPF 411
PBP5efam
AQKTAFDSLGGKAGSTVATTPKTGDLLALASSPSYDPNKMTNGISQEDYKSYEEN
PEQPF 405
PBP2a
VQKSIYNNMKNDYGSGTAIHPQTGELLALVSTPSYDVYPFMYGMSNEEYNKLTED
KKEPL 391
= . = . * . = ** *. **** ***** = **===* == *=
. . . . . . . . . . . . .
PBP5Efas
TARFATGYAPGSTFKIITGAIGLDAGTLKPDEELEINGLKWQKDKSWGGYFAT
RVKEAS- 470
PBP5efam
ISRFATGYAPGSTFKMITAAIGLDNGTIDPNEVLTINGLKWQICDSSWGSYQVTR
VSDVS- 464
PBP2a
LNKFQITTSPGSTQKILTAMIGLNNKTLDDKTSYKIDGKGWQKDKSWGGYNVTRY
EVVNG 451
=* =**** *==* ***. *. *.* **** ***
* **
. . . .
34
CA 02770771 2012-02-10
PBP5Efas
PVNLRTALVNSDNIYFAQQTLRMGEDKFRAGLNKFIFGEELDLPIAMTPAQISNEDK
FNS 530
PBP5efam
QVDLKTALIYSDNIYTAQETLKMGEKKFRIGLDKFIFGEDLDLPISMNPAQISNEDSF
NS 524
PBP2a
NIDLKQAIESSDNIFFARVALELGSKKFEKGMKKLGVGEDIPSDYPFYNAQISNKN-
LDN 510
==*= *= ****= *= =* .* ** *. *. **== . *****.=
. . . . . . . .
PBP5Efas
EILLADTGYGQGQLLISPIQQATMYSVFQNNGTLVYPKLVLDKETKK-
KDNVISANAANT 589
PBP5efam
DILLADTGYGQGELLINPIQQAAMYSVFANNGTLVYPKLIADKETKD-
KKNVIGETALQT 583
PBP2a
EILLADSGYGQGEILINPVQILSIYSALENNGNINAPHLLKDTKNKVWKKNIISKENI
NL 570
. = =**********..** ** ..** = *** = *=*= * = * * *=* =
. . . . . . . . . .
PBP5Efas
IATDLLGSVEDPSGYVYNMYNPNFSLAAKTGTAEIKDKQDTDGKENSFLLTLDRSN
NKFL 649
PBP5efam
IVPDLREVVQDVNGTAHSLSALGIPLAAKTGTAEIKEKQDVKGKENSFLFAFNPDN
QGYM 643
PBP2a LNDGMQQVVN--
KTHKEDIYRSYANLIGKSGTAELKMKQGESGRQIGWFISYDKDNPNMM 628
= = *= = * *=****=* ** *== * .
. . . . . . . . ..... . . .
CA 02770771 2012-02-10
PBP5Efas TMIMVENSGENGSATDISKPLIDYLEATIK ------------- 679
PBP5efam MVSMLENKEDDDSATKRASELLQYLNQNYQ ---------------- 673
PBP2a MAINVKDVQDKGMASYNAKISGKVYDELYENGNKKYDIDE 668
. . . .
The alignment was performed with sequences corresponding to PBP5 from the
enterococci E. faecalis (Efas) and E. faecium (Efam) with MRSA PBP2a. Marked
sequences (bold - PBP5; underlined - PBP2a) correspond to the used PBP2a
region to
generate monoclonal antibodies. Amino acids corresponding to the enzyme active
core
are marked in italic.
Thus, in vitro protection assays, determination of lethal dose and LD50, and
in vivo
assays (sublethal dose with renal quantification and survival assay with
lethal dose)
with an enterococcus strain in murine model (Balb/C mice) were conducted, as
previously conducted with MRSA. These results can be seen in the corresponding
reports.
1.1. In vitro protection assays
The objective of this assay was to evaluate the in vitro protection conferred
by the
antibody against VRE enterococcus strain.
In vitro protection test (MIC), clone 38 purified monoclonal antibody
(90/DA5/CB5/AA3 hib 77) against Enterococcus f clinical strain (VRE), Richet
laboratory.
Conditions:
Sample: purified from supernatant by HPLC, SelecSure MAB resin, dialysed,
lyophilized.
Antibody quantification (Lowry's method): 3.5 mg/mL
Inoculum: VRE strain
36
CA 02770771 2012-02-10
Pre-inoculum: 1 VRE colony in 20 mL Lb and vancomycin (10 mg/mL), ON 37 C, 160
rpm
Inoculum: 400 mL of pre-inoculum in 20 mL Lb, 200-mL erlenmayer, 37 C, 160 rpm
DO600nm reading after 7 hours: 0.7
Quantification: 5.5 x 108 bacteria/mL
Test conditions:
Inoculum: 5.5 x 105 bacteria
Antibody concentrations: 300, 400, 500, 600, and 700 mg of antibody
Culture medium: 1 mL of Luria broth
Cell culture plate, 24 cavities
Positive control: Luria broth + bacterial inoculum
Negative control: Luria broth
Incubation: 18 hours, 37 C
Figure 11 shows the result of the evaluation of the protection conferred by
the
antibody. In Figure 11, we have:
A. 300 mg of antibody
B. 400 mg of antibody
C. 500 mg of antibody
D. 600 mg of antibody
E. Negative control
F. Positive control
G. 700 mg of antibody
1.2. LD50 and lethal dose determination by intraperitoneal route - vancomycin-
resistant Enterococcus faecium
Protocol:
Female 7-week Balb/C animals, mean weight of 20 grams
Day 01 - pre-inoculum: 1 VRE strain colony in 10 mL of Lb broth and vancomycin
(10
mg/mL), 50-mL Falkow tube, growth ON 37 C, 160 rpm
37
CA 02770771 2012-02-10
Day 02 - inoculum: 1 mL of pre-inoculum in 50 mL of Lb broth/vancomycin (250-
mL
erlenmayer) - 4 vials, 37 C, 160 rpm, growth until OD600nm = 0.80
centrifugation for 10 min, 4000 rpm, resuspension in PBS lx sterile, OD 1.2
Quantification: 2.1 x 108 bacteria/mL
A. 60 microliters (1.5 x 107)
B. 300 microliters (6.5 x 107)
C. 900 microliters (reduced to 300 microliters/dose) (1.5 x 108)
D. 4.5 mL (reduced to 300 microliters/dose) (6.5 x 108)
E. 9.0 mL (1.2 x 109 bacteria) (reduced to 300 microliters/dose)
F. 45.0 mL (6.5 x 109 bacteria) reduced to 300 microliters/dose)
The animal observation was performed from day 02 to the tenth day of the
assay.
The result is shown in Figure 12, and it is concluded that the lethal dose is
1.2 x 109
bacteria and LD50 is 6.5 x 108 bacteria.
Renal quantification of animals surviving in the seventh day:
A (1.5 x 107): no bacterial growth
B (6.5 x 107): no bacterial growth
C (1.5 x 108): 3100 bacteria
D (6.5 x 108): 2.8 x 104 bacteria
1.3. In vivo protection test - survival test - lethal dose, systemic infection
by
intraperitoneal route in murine model
The objective of this assay was to evaluate the efficacy of in vivo protection
for anti-
PBP2a monoclonal antibody against systemic infection with lethal dose,
Enterococcus
faecium (VRE) strain.
1. Antibody (purified from supernatant of the cell culture in medium with
serum)
- Dialysed and lyophilized purified sample (HPLC SelecSure MAB), resuspended
and
filtered before use.
- Quantification (Lowry's method): 1.0 mg/mL
38
CA 02770771 2012-02-10
2. Murine model: female, 8-week Balb/C animals, weigh from 23 to 25 grams
3. Protocol:
group A (6 animals): 650 mg of antibody (350 mg + 300 mg)
group B (6 animals): control (saline administration)
4. Preparation of bacterial inoculum:
VRE strain:
pre-inoculum, day 01, 10 mL of BHI broth and vancomycin at 10 mg/mL ON, 37 C,
160 rpm
inoculum, day 03: 300 mL of pre-inoculum in 30 mL of BHI and vancomycin, D0600
of
1.31, centrifuged for 10 min, 4,000 rpm, resuspended in sterile PBS 0.5x,
adjustment at
OD = 1.10, dilutions and plates for quantification (2.0 x 108 bacteria/mL);
inoculum: 12
mL, centrifugate resuspended in 300 mL, IP route (-2.2 x 109 bacteria)
Time schedule:
Day 01: IP inoculation of antibody (350 mg)
Day 02: IP inoculation of antibody (300 mg), systemic infection in the
afternoon (IP,
250 mL of bacterial solution -2.2 x 109 bacteria)
Day 02 to Day 13: animal observation.
The results are shown in Figure 13. Only 2 treated animals died (66.6% of
protection). All control animals died in the second day.
1.4. In vivo protection test - systemic infection by intraperitoneal route in
murine
model with vancomycin-resistant Enterococcus faecium
The objective was to evaluate the in vivo protection efficacy of anti-PBP2a
and PBP5
monoclonal antibody from enterococci against systemic infection, E. faecium
(VRE)
strain.
Test:
1. Antibody (purified from supernatant of the cell culture in medium with
serum)
- Dialysed, lyophilized, and resuspended purified sample (AffiPrep ProteinA
Biorad/HPLC SelecSure MAB).
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CA 02770771 2012-02-10
- Quantification (Lowry's method): 1.5 mg/mL
2. Murine model: female, 8-week Balb/C animals, weigh from 19 to 23 grams
3. Protocol:
group A (4 animals): 500 micrograms of antibody (in 2 months, d01, d02)
group B (4 animals): non-protected control
4. Preparation of bacterial inoculum:
Iberian MRSA strain:
pre-inoculum, 10 mL of Lb broth ON, 37 C, 120 rpm
inoculum: 200 microliters of pre-inoculum in 20 mL of Lb, D0600 of 0.80,
centrifuged
for 10 min, 4,000 rpm, resuspended in sterile PBS 0.5x, adjustment at OD =
0.51,
dilutions and plates for quantification (2.4 x 108 bacteria/mL); inoculum: 500
microliters, IP route (2.4 x 108 bacteria)
Time schedule:
Day 01: inoculation of 250 micrograms of antibody by intraperitoneal route
Day 02: inoculation of 250 micrograms of antibody by intraperitoneal route and
systemic infection (intraperitoneal, 500 microliters of bacterial solution)
Day 06: euthanasia, bacterial quantification in kidneys
Based on the results above, we can highlight that:
In vitro protection assays - determination of minimum inhibitory
concentration:
A total of 700 micrograms of antibody is able to block the growth of 550,000
bacteria. These values are higher than the MIC obtained for MRSA strains,
which were
approximately 500 micrograms.
In vivo protection assays:
In vivo protection test - systemic infection by intraperitoneal route in
murine model
with sublethal dose of vancomycin-resistant Enterococcus faecium.
The animals received 500 micrograms of monoclonal antibody, intraperitoneal
route
(IP), and were subjected to systemic infection, IP route, with 2.4 x 108
bacteria. Four
days after, they were subjected to euthanasia and excision of kidneys for
bacterial
CA 02770771 2012-02-10
quantification. Treated animals presented a mean of 87.5 bacteria/animal,
while controls
(non-treated infected animals) presented a mean of 211,000 bacteria/animal.
Survival test after lethal dose administration
The animals received 650 micrograms of monoclonal antibody (IP route) and were
subjected to systemic infection (IP route) and daily observed for 10 days.
Control (non-
treated) animals died until the second day after the infection; two of the
treated animals
(6) died in the second day; the others remained alive until the end of the
trial. Survival
rate was 66.6%.
Therefore, once more we have confirmed that MRSA anti-PBPa monoclonal antibody
showed to confer cross protection against enterococci. However, the doses
needed for
conferring protection were higher than the ones used against MRSA in similar
conditions. This is probably due to the lower capacity of the antibody to
recognize
PBP5 with the same efficacy as PBP2a, which it was developed for.
Therefore, the current invention described here - anti-PBP2a monoclonal
antibodies,
able to specifically bind to PBP2a and homologous sequences - has
applicability in
infections caused by bacteria presenting this protein or similar substances
(MRSA,
MRSE, and Enterococcus spp., and any other pathogen that has a protein
homologous
to PBP2a).
It is important to emphasize that once these infections are a worldwide
problem and
the assays were conducted against the main known epidemic MRSA clones, the
product
of the current invention has application in any place where there are
infections by this
pathogen.
The documents relative to the state of the art of knowledge of the inventors,
cited in
the current descriptive report, are listed below.
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