U.S. patent application number 11/374065 was filed with the patent office on 2006-10-05 for staphylococcal immunotherapeutics via donor selection and donor stimulation.
Invention is credited to Timothy J. Foster, Magnus Hook, Joseph M. Patti.
Application Number | 20060222651 11/374065 |
Document ID | / |
Family ID | 26794752 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060222651 |
Kind Code |
A1 |
Patti; Joseph M. ; et
al. |
October 5, 2006 |
Staphylococcal immunotherapeutics via donor selection and donor
stimulation
Abstract
A method and composition for the passive immunization of
patients infected with or susceptible to infection from
Staphylococcus bacteria such as S. aureus and S. epidermidis
infection is provided that includes the selection or preparation of
a donor plasma pool with high antibody titers to carefully selected
Staphylococcus adhesins or MSCRAMMs, or fragments or components
thereof, or sequences with substantial homology thereto. The donor
plasma pool can be prepared by combining individual blood or blood
component samples which have higher than normal titers of
antibodies to one or more of the selected adhesins or other
proteins that bind to extracellular matrix proteins, or by
administering carefully selected proteins or peptides to a host to
induce the expression of desired antibodies, and subsequently
recovering the enhanced high titer serum or plasma pool from the
treated host.
Inventors: |
Patti; Joseph M.; (Cumming,
GA) ; Foster; Timothy J.; (Dublin, IE) ; Hook;
Magnus; (Houston, TX) |
Correspondence
Address: |
STITES & HARBISON PLLC
1199 NORTH FAIRFAX STREET
SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
26794752 |
Appl. No.: |
11/374065 |
Filed: |
March 14, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10091494 |
Mar 7, 2002 |
7045131 |
|
|
11374065 |
Mar 14, 2006 |
|
|
|
09386960 |
Aug 31, 1999 |
6692739 |
|
|
10091494 |
Mar 7, 2002 |
|
|
|
60098449 |
Aug 31, 1998 |
|
|
|
Current U.S.
Class: |
424/165.1 ;
530/388.4 |
Current CPC
Class: |
C07K 16/1271 20130101;
C07K 2317/76 20130101; C07K 2317/20 20130101; A61K 2039/505
20130101; A61K 9/0019 20130101; A61K 39/00 20130101 |
Class at
Publication: |
424/165.1 ;
530/388.4 |
International
Class: |
A61K 39/40 20060101
A61K039/40; C07K 16/12 20060101 C07K016/12 |
Claims
1. A method of obtaining a human immunoglobulin composition having
a higher antibody titer to a staphylococcal clumping factor A
(ClfA) adhesin than that found in pooled intravenous immunoglobulin
obtained from unselected human donors comprising obtaining blood or
plasma samples from human donors, identifying those blood or plasma
samples from high-titer donors having the presence of an antibody
titer to ClfA in an amount which is higher than that found in
pooled intravenous immunoglobulin obtained from unselected donors,
recovering blood or plasma from the identified high-titer donors,
and treating the donor blood or plasma to obtain a human
immunoglobulin composition in a purified state that has an antibody
titer to ClfA in an amount which is higher than that found in
intravenous immunoglobulin obtained from unselected donors.
2. The method according to claim 1 wherein donors are identified
which have an antibody titer to ClfA in an amount which is 2-fold
or greater than that found in pooled intravenous immunoglobulin
obtained from unselected donors.
3. The method according to claim 1 wherein donors having a high
titer to ClfA are determined by identifying those samples having a
high titer of antibodies to the A domain of ClfA.
4. The method according to claim 1 further comprising identifying
those samples also having an antibody titer to a second
staphylococcal adhesin which is higher than that found in pooled
intravenous immunoglobulin obtained from unselected donors.
5. The method according to claim 4 wherein the second
staphylococcal adhesin is an Sdr protein.
6. The method according to claim 5 wherein donors having a high
titer to the Sdr protein are determined by identifying those
samples having a high titer of antibodies to the A domain of the
Sdr protein.
7. The method according to claim 5 wherein the Sdr protein is
selected from the group consisting of SdrF, SdrG, and SdrH.
8. A human immunoglobulin composition obtained by the method of
claim 1.
9. A method of obtaining a human immunoglobulin composition having
a higher antibody titer to a staphylococcal ClfA adhesin than that
found in pooled intravenous immunoglobulin obtained from unselected
human donors comprising administering ClfA to a host donor in an
amount sufficient so as to induce an antibody titer to ClfA in an
amount which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors, recovering blood or
plasma from the host donor, and treating the donor blood or plasma
to obtain a human immunoglobulin composition in a purified state
that has an antibody titer to ClfA which is higher than that found
in pooled intravenous immunoglobulin obtained from unselected
donors.
10. The method according to claim 9 wherein the host donor is
induced to have an antibody titer to ClfA in an amount which is
higher than that found in pooled intravenous immunoglobulin
obtained from unselected donors by administering the A domain of
ClfA to the host donor an amount sufficient so as to induce an
antibody titer to ClfA in an amount which is higher than that found
in pooled intravenous immunoglobulin obtained from unselected
donors.
11. The method according to claim 9 wherein immunoglobulin is
obtained that has an antibody titer to ClfA in an amount which is
2-fold or greater than that found in pooled intravenous
immunoglobulin obtained from unselected donors.
12. The method according to claim 9 further comprising
administering a second staphylococcal adhesin to a host donor in an
amount sufficient so as to induce an antibody titer to the second
adhesin in an amount which is higher than that found in pooled
intravenous immunoglobulin obtained from unselected donors.
13. The method according to claim 12 wherein the second
staphylococcal adhesin is an Sdr protein.
14. The method according to claim 13 wherein the host donor is
induced to have an antibody titer to the Sdr protein in an amount
which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors by administering the
A domain of the Sdr protein an amount sufficient so as to induce an
antibody titer to the Sdr protein in an amount which is higher than
that found in pooled intravenous immunoglobulin obtained from
unselected donors.
15. The method according to claim 13 wherein the Sdr protein is
selected from the group consisting of SdrF, SdrG, and SdrH.
16. A human immunoglobulin composition obtained by the method of
claim 9.
17. A method of obtaining a human immunoglobulin composition having
a higher than normal antibody titer to a staphylococcal Sdr protein
comprising obtaining blood or plasma samples from donors,
identifying those blood or plasma samples from high-titer donors
having the presence of an antibody titer to a staphylococcal Sdr
protein in an amount which is higher than that found in pooled
intravenous immunoglobulin obtained from unselected donors,
recovering blood or plasma from the identified high-titer donors,
and treating the donor blood or plasma to obtain a human
immunoglobulin composition in a purified state that has an antibody
titer to a staphylococcal Sdr protein in an amount which is higher
than that found in intravenous immunoglobulin obtained from
unselected donors.
18. The method according to claim 17 wherein donors are identified
which have an antibody titer to a staphylococcal Sdr protein in an
amount which is 2-fold or greater than that found in pooled
intravenous immunoglobulin obtained from unselected donors.
19. The method according to claim 18 wherein donors having a high
titer to a staphylococcal Sdr protein are determined by identifying
those samples having a high titer of antibodies to the A domain of
a staphylococcal Sdr protein.
20. The method according to claim 18 further comprising identifying
those samples also having an antibody titer to a second adhesin
which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors.
21. The method according to claim 20 wherein the second adhesin is
also a staphylococcal Sdr protein.
22. The method according to claim 21 wherein donors having a high
titer to the second staphylococcal Sdr protein are determined by
identifying those samples having a high titer of antibodies to the
A domain of the second staphylococcal Sdr protein.
23. A human immunoglobulin composition obtained by the method of
claim 18.
24. A method of obtaining a human immunoglobulin composition having
a higher than normal antibody titer to a staphylococcal Sdr protein
comprising administering a staphylococcal Sdr protein to a host
donor in an amount sufficient so as to induce an antibody titer to
a staphylococcal Sdr protein in an amount which is higher than that
found in pooled intravenous immunoglobulin obtained from unselected
donors, recovering blood or plasma from the host donor, and
treating the donor blood or plasma to obtain a human immunoglobulin
composition in a purified state that has an antibody titer to a
staphylococcal Sdr protein which is higher than that found in
pooled intravenous immunoglobulin obtained from unselected
donors.
25. The method according to claim 24 wherein the host donor is
induced to have an antibody titer to a staphylococcal Sdr protein
in an amount which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors by administering the
A domain of a staphylococcal Sdr protein to the host donor in an
amount sufficient so as to induce an antibody titer to a
staphylococcal Sdr protein in an amount which is higher than that
found in pooled intravenous immunoglobulin obtained from unselected
donors.
26. The method according to claim 24 wherein immunoglobulin is
obtained that has an antibody titer to a staphylococcal Sdr protein
in an amount which is 2-fold or greater than that found in pooled
intravenous immunoglobulin obtained from unselected donors.
27. The method according to claim 24 further comprising
administering a second adhesin to a host donor in an amount
sufficient so as to induce an antibody titer to the second adhesin
in an amount which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors.
28. The method according to claim 27 wherein the second adhesin is
a second staphylococcal Sdr protein.
29. The method according to claim 28 wherein the host donor is
induced to have an antibody titer to the second staphylococcal Sdr
protein in an amount which is higher than that found in pooled
intravenous immunoglobulin obtained from unselected donors by
administering the A domain of the second staphylococcal Sdr protein
an amount sufficient so as to induce an antibody titer to the
second staphylococcal Sdr protein in an amount which is higher than
that found in pooled intravenous immunoglobulin obtained from
unselected donors.
30. A human immunoglobulin composition obtained by the method of
claim 24.
31. A method of obtaining an immunoglobulin composition having a
higher antibody titer to a staphylococcal clumping factor A (ClfA)
adhesin than that found in pooled intravenous immunoglobulin
obtained from unselected human donors comprising obtaining blood or
plasma samples from human donors, and: (a) identifying those blood
or plasma samples from high-titer donors having the presence of an
antibody titer to ClfA in an amount which is higher than that found
in pooled intravenous immunoglobulin obtained from unselected
donors and identifying those samples also having an antibody titer
to a second staphylococcal adhesin selected from the group
consisting of a fibronectin binding protein, a collagen binding
protein, a fibrinogen binding protein, an elastin binding protein
and an MHC-II analogous protein in an amount which is higher than
that found in pooled intravenous immunoglobulin obtained from
unselected donors. (b) recovering blood or plasma from the
identified high-titer donors, and (c) treating the recovered blood
or plasma to obtain immunoglobulin in a purified state that has an
antibody titer to ClfA in an amount which is higher than that found
in pooled intravenous immunoglobulin obtained from unselected
donors and an antibody titer to the second staphylococcal adhesin
in an amount which is higher than that found in pooled intravenous
immunoglobulin obtained from unselected donors;
32. The method according to claim 31 wherein the second
staphylococcal adhesin is selected from the group consisting of
proteins fibronectin binding protein A (FnBP-A), fibronectin
binding protein B (FnBP-B), clumping factor protein B (ClfB), SdrC,
SdrD, SdrE, SdrF, SdrG, SdrH, CNA, and EbpS.
33. The method of claim 31 wherein donors having a high titer to
the staphylococcal Sdr protein are determined by identifying those
samples having a high titer of antibodies to the A domain of the
staphylococcal Sdr protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 10/091,494, filed Mar. 7, 2002, which was a divisional
application of U.S. Ser. No. 09/386,960, filed Aug. 31, 1999, now
U.S. Pat. No. 6,692,739, which claims the benefit of provisional
application U.S. Ser. No. 60/098,449, filed Aug. 31, 1998.
FIELD OF THE INVENTION
[0002] The invention is in the field of biological products for the
treatment, prevention and diagnosis of bacterial infections.
BACKGROUND OF THE INVENTION
[0003] The staphylococci are Gram-positive spherical cells, usually
arranged in grape-like irregular clusters. Some are members of the
normal flora of the skin and mucous membranes of humans, others
cause suppuration, abscess formation, a variety of pyogenic
infections, and even fatal septicemia. Pathogenic staphylococci
often hemolyze blood, coagulate plasma, and produce a variety of
extracellular enzymes and toxins. The most common type of food
poisoning is caused by a heat-stable staphylococci enterotoxin.
[0004] The genus Staphylococcus has at least 30 species. Three main
species of clinical importance are Staphylococcus aureus,
Staphylococcus epidermidis, and Staphylococcus haemolyticus.
Staphylococcus aureus is coagulase-positive, which differentiates
it from the other species. S. aureus is a major pathogen for
humans. Almost every person has some type of S. aureus infection
during a lifetime, ranging in severity from food poisoning or minor
skin infections to severe life-threatening infections. The
coagulase-negative staphylococci are normal human flora which
sometimes cause infection, often associated with implanted devices,
especially in very young, old and immunocompromised patients.
Approximately 75% of the infections caused by coagulase-negative
staphylococci are due to parasitic S. epidermidis. Infections due
to Staphylococcus haemolyticus, Staphylococcus hominis, and other
species are less common. S. saprophyticus is a relatively common
cause of urinary tract infections in young women.
[0005] Staphylococcus bacteria such as S. aureus thus cause a
spectrum of infections that range from cutaneous lesions such as
wound infections, impetigo, and furuncles to life-threatening
conditions that include pneumonia, septic arthritis, sepsis,
endocarditis, and biomaterial related infections. S. aureus
colonization of the articular cartilage, of which collagen is a
major component, within the joint space appears to be an important
factor contributing to the development of septic arthritis.
Hematogenously acquired bacterial arthritis remains a serious
medical problem. This rapidly progressive and highly destructive
joint disease is difficult to eradicate. Typically less than 50% of
the infected patients failing to recover without serious joint
damage. S. aureus is the predominant pathogen isolated from adult
patients with hematogenous and secondary osteomyelitis.
[0006] In hospitalized patients, Staphylococcus aureus is a major
cause of infection. Initial localized infections of wounds or
indwelling medical devices can lead to more serious invasive
infections such as septicemia, osteomyelitis, mastitis and
endocarditis. In infections associated with medical devices,
plastic and metal surfaces become coated with host plasma and
matrix proteins such as fibrinogen and fibronectin shortly after
implantation. The ability of Staphylococcus bacteria such as S.
aureus to adhere to these proteins is essential to the initiation
of infection. Vascular grafts, intravenous catheters, artificial
heart valves, and cardiac assist devices are thrombogenic and prone
to bacterial colonization. S. aureus is the most damaging pathogen
of such infections, and other Staphylococci bacteria such as S.
epidermidis are also responsible for a significant amount of
dangerous infections, particularly those associated with implanted
devices.
[0007] There is a strong and rapidly growing need for therapeutics
to treat infections from Staphylococcus bacteria such as S. aureus
and S. epidermidis infections which are effective against
antibiotic resistant strains of the bacteria. The U.S. National
Institutes for Health has recently indicated that this goal is now
a national priority.
MSCRAMMs
[0008] The successful colonization of the host is a process
required for most microorganisms to cause infections in animals and
humans. Microbial adhesion is the first crucial step in a series of
events that can eventually lead to disease. Pathogenic
microorganisms colonize the host by attaching to host tissues or
serum conditioned implanted biomaterials, such as catheters,
artificial joints, and vascular grafts, through specific adhesins
present on the surface of the bacteria. MSCRAMMs (Microbial Surface
Components Recognizing Adhesive Matrix Molecules) are a family of
cell surface adhesins that recognize and specifically bind to
distinct components in the host's extracellular matrix. Once the
bacteria have successfully adhered to and colonized host tissues,
their physiology is dramatically altered and damaging components
such as toxins and proteolytic enzymes are secreted. Moreover,
adherent bacteria often produce a biofilm and quickly become more
resistant to the killing effect of most antibiotics.
[0009] For example, S. aureus is known to express a repertoire of
different MSCRAMMs that can act individually or in concert to
facilitate microbial adhesion to specific host tissue components.
MSCRAMMs provide an excellent target for immunological attack by
antibodies. The presence of the appropriate anti-MSCRAMM high
affinity antibodies has a double-edged attack, first the antibodies
prevent microbial adherence and second the increased titers of
MSCRAMM antibodies facilitate a rapid clearance of the organism
from the body through bacterial lysis, opsonization, phagocytosis
and complement activation.
Passive Immunization to Bacterial Infections
[0010] Immunoglobulins (A, D, E, G, and M) are used by the body as
a primary defense to infections. Complement, available as a
precursor protein which is activated by the presence of
microorganisms and globulins, also exhibits antibacterial
activities. After previous antigenic exposure, the immune system
produces a series of globulins which attach to and coat bacteria or
neutralize viruses so that they are readily recognized,
phagocytized and destroyed by neutrophils and macrophages. Foreign
proteins of invading organisms also stimulate a humoral immune
response which over a period of time from three to six weeks
amplifies the number of cells designed to recognize and destroy
specific invaders.
[0011] In the last decade, intravenous immunoglobulin (IVIG)
therapy has become a major treatment regime for bacterial
infections, especially in immunocompromised patients (Siber, New
Eng. J. Med., 327:269-271, 1992). IVIG therapy has exhibited
efficacy against more than thirty-five diseases caused by
immunopathologic mechanisms. Passive immunization against
infections has been particularly successful with immune globulins
specific for tetanus, hepatitis B, rabies, chicken pox and
cytomegalovirus. There has been an inconsistent and disappointing
response to the use of immunoglobulins to prevent nosocomial
infections, likely due to the variety of strains of bacteria found
in hospitals and the emergence of new serotypes. Passive
immunization requires the presence of high and consistent titers of
antibodies to the infecting pathogens.
[0012] Supplemental immunoglobulin therapy has been shown to
provide some measure of protection against certain encapsulated
bacteria such as Hemophilus influenzae and Streptococcus
pneumoniae. Infants who are deficient in antibody are susceptible
to infections from these bacteria and bacteremia and sepsis are
common. When anti-Streptococcal and anti-Hemophilus antibodies are
present, they provide protection by promoting clearance of the
respective bacteria from the blood. In the case of antibody to
Staphylococcus, the potential use of supplemental immunoglobulin to
prevent or treat infection has been much less clear.
[0013] Early attempts to treat Staphylococcus infections focused on
the potential use of supplemental immunoglobulin to boost
peritoneal defenses, such as opsonic activity, in patients
receiving continuous ambulatory peritoneal dialysis. Standard
intravenous immunoglobulin (IVIG) was shown to have lot to lot
variability for opsonic activity to S. epidermidis (L. A. Clark and
C. S. F. Easmon, J. Clin. Pathol. 39:856 (1986)). In this study,
one third of the IVIG lots tested had poor opsonization with
complement, and only two of fourteen were opsonic without
complement. Thus, despite the fact that the IVIG lots were made
from large plasma donor pools, good opsonic antibody to S.
epidermidis was not uniformly present. Moreover, this study did not
examine whether IVIG could be used to prevent or treat S.
epidermidis infections or bacterial sepsis.
[0014] Prior studies have associated coagulase-negative
Staphylococcus bacteria, such as S. epidermidis, as the most common
species causing bacteremia in neonates receiving lipid emulsion
infusion (Freeman, J. et al., N. Engl. J. Med. 323:301, 1990).
These neonates had low levels of opsonic antibody to S. epidermidis
despite the fact that the sera had clearly detectable levels of IgG
antibodies to S. epidermidis peptidoglycan (Fleer, A. et al., J.
Infect. Dis. 2:426, 1985). This was surprising because
anti-peptidoglycan antibodies were presumed to be the principal
opsonic antibodies. Thus, while suggesting that neonatal
susceptibility to S. epidermidis might be related to impaired
opsonic activity, these studies also suggested that many antibodies
directed against S. epidermidis are not opsonic and would not be
capable of providing protection when given passively to
neonates.
[0015] In addition, an antigen binding assay was used to analyze
IgG antibody to S. epidermidis in patients with uncomplicated
bacteremia and those with bacteremia and endocarditis (F. Espersen
et al., Arch. Intern. Med. 147:689 (1987)). This assay used an
ultrasonic extract of S. epidermidis to identify S. epidermidis
specific IgG. None of the patients with uncomplicated bacteremia
had IgG antibodies to S. epidermidis. These data suggest that IgG
does not provide effective eradication of S. epidermidis from the
blood. In addition, 89% of bacteremic patients with endocarditis
developed high levels of IgG to S. epidermidis. In these patients,
IgG was not protective since high levels of IgG antibody were
associated with serious bacteremia and endocarditis. Based on these
studies, the protective role of IgG in S. epidermidis sepsis and
endocarditis was questionable, especially in the presence of
immaturity, debilitation, intralipid infusion, or
immunosuppression.
[0016] Animal studies in the literature that demonstrated
immunoglobulin protection against Staphylococcus infections have
shown strain specificity by enzyme-linked immunosorbent assays
(ELISA) and have utilized normal adult mice in protection studies.
Animal models typically have used mature animals with normal
immunity with unusually virulent strains or overwhelming-challenge
doses of bacteria. Human patients are generally immunologically
immature or debilitated. Human patients also get somewhat indolent
infections with low virulence pathogens such as S. epidermidis with
death usually attributable to secondary complications. Models that
have used unusual strains or overwhelming bacterial doses,
generally induce rapid fulminant death. These are important factors
since antibodies generally work in concert with the host cellular
immune system (neutrophils, monocytes, macrophages and fixed
reticuloendothelial system). The effectiveness of antibody therapy
may therefore be dependent on the functional immunologic
capabilities of the host. To be predictive, animal models must
closely emulate the clinical condition in which the infection would
occur and capture the setting for therapy. Moreover, the animal
studies have yielded inconsistent results.
[0017] One model has been reported which used an unusually virulent
strain of S. epidermidis. Infected-mature mice developed 90 to 100%
mortality within 24 to 48 hours (K. Yoshida et al., Japan. J.
Microbiol. 20:209 (1976)). Antibody to S. epidermidis surface
polysaccharide was protective in these mice. Protection was shown
to occur with an IgM fraction, but not the IgG fraction (K. Yoshida
and Y. Ichiman, J. Med. Microbiol. 11:371 (1977)). This model,
however, presents a pathology which is very different from that
seen in typically infected patients. Intraperitoneally-challenged
mice developed symptoms of sepsis within minutes of receiving the
injection and died in 24 to 48 hours. This particular pathology is
not observed in Staphylococcus infected humans. The highly virulent
strain of S. epidermidis may represent an atypical type of
infection. moreover, isolates of S. epidermidis from infected
humans did not kill mice in this model.
[0018] In 1987, these animal studies were extended to include the
evaluation of antibodies in human serum against selected virulent
strains of S. epidermidis (Y. Ichiman et al., J. Appl. Bacteriol.
63:165 (1987)). In contrast to the previous data, protective
antibody was found in the IgA, IgM and IgG immunoglobulin
fractions. A definitive role for any single class of immunoglobulin
(IgG, IgM, IgA) could not be established.
[0019] In this animal model, normal adult mice were used and
mortality was determined. Death was considered to be related to the
effect of specific bacterial toxins, not sepsis (K. Yoshida et al.,
Japan J. Microbiol. 20:209 (1976)). Most clinical isolates did not
cause lethal infections, and quantitative blood cultures were not
done. Moreover, this study provided little insight as to whether
antibody could successfully prevent or treat S. epidermidis sepsis
in immature or immunosuppressed patients.
[0020] In a later study, serotype specific antibodies directed
against S. epidermidis capsular polysaccharides were tested in the
animal model. Results showed that serotype-specific antibodies were
protective, but that each antibody was directed against one
serotype as measured by ELISA. Protection was equally serotype
specific. Protection against heterologous strains did not occur. In
addition, it was concluded that protection was afforded by the IgM
antibody.
[0021] There has been no compelling evidence that IVIG would be
effective to treat S. epidermidis infections or sepsis,
particularly where the patients are immature or immune suppressed
or where multiple S. epidermidis serotypes are involved. Thus, for
example, a recent and extensive review of the pathogenesis,
diagnosis, and treatment of S. epidermidis infections does not
include immunoglobulin as a potential prophylactic or therapeutic
agent (C. C. Patrick, J. Pediatr. 116:497 (1990)). In addition,
there have been no U.S. patents which describe the effective use of
IVIG therapy in conjunction with antibodies to MSCRAMMs such as
described above.
[0022] U.S. Pat. No. 5,505,945 discloses compositions for passive
immunity that contain a full repertoire of immunoglobulins,
including IgA, IgM, and IgG to combat infections from
microorganisms and viruses at wound, surgical, or burn sites. The
compositions contain elevated antibody titers for several
pathogens, including S. aureus, Coagulase Negative Staphylococci
Enterococci, S. epidermidis, P. aeruginose, E. coli, and
Enterobacter spp. However, these compositions are specifically
designed to avoid the use of intravenous immunoglobulin or IVIG
therapy, and instead are applied in the form of ointments, creams,
sprays and the like which are designed for topical application
only.
[0023] U.S. Pat. No. 4,717,766 discloses a method of preparing high
titer anti-respiratory syncytial virus intravenous
immunoglobulins.
[0024] U.S. Pat. No. 5,219,578 describes a composition and method
for immunostimulation in mammals, and specifically describes the
isolation of an IgG fraction from goats free from foreign or
artificially induced antigens and the utilization of the isolated
immunoglobulins fraction to induce a stimulated immune
response.
[0025] U.S. Pat. No. 5,548,066 describes a method for drawing blood
from a donor animal, permitting blood to clot, separating liquid
from cellular material, and then clarifying, concentrating and
sterilizing the product.
[0026] U.S. Pat. No. 4,412,990 discloses an intravenous
pharmaceutical composition containing immunoglobulin (IgG) and
fibronectin that exhibits a synergistic opsonic activity which
results in enhanced phagocytosis of bacteria, immune complexes and
viruses.
[0027] U.S. Pat. No. 4,994,269 discloses the topical use of
monoclonal antibodies for the prevention and treatment of
experimental P. aeriginosa lung infections. Specifically, the
antibodies are administered via aerosol spray to the lungs. Results
show beneficial effects in the treatment of affected patients.
[0028] U.S. Pat. No. 4,714,612 discloses the use of a non-specific
gamma globulin IgG in a mouthwash for the prevention of gingivitis.
Another mouthwash with monoclonal antibodies is described by Ma et
al. in Arch. Oral Biol., 35 Supp: 115S-122S, in 1990. The
monoclonal antibodies were specific for Streptococcus mutans, and
patients treated with the mouthwash remained free of S. mutans for
up to two years. Those who did not take the mouthwash experiences
recolonization of S. mutans within two days.
[0029] U.S. Pat. Nos. 5,718,889 and 5,505,945 describe the direct,
concentrated local delivery of passive immunity which is
accomplished by applying a composition having a full repertoire of
immunoglobulins (IgG, IgM and IgA) to biomaterials, implants,
tissues, and wound and burn sites.
[0030] U.S. Pat. No. 5,571,511 describes the use of immunoglobulin
from individual samples or pools of serum, plasma, whole blood, or
tissue for the treatment of a Staphylococcus infection.
Immunoglobulin is identified by performing a first assay to
identify immunoglobulin which is reactive with a preparation of a
first Staphylococcus organism, performing a second assay to
identify immunoglobulin which is reactive with a preparation of a
second Staphylococcus organism, and selecting immunoglobulin which
is reactive with the preparations from both the first and second
Staphylococcus organisms. Reactivity is determined in immunological
assays which may be binding assays, opsonization assays, or
clearance assays. Preferably, the preparations of the first and the
second Staphylococcus organisms are derived from different
serotypes or different species, such as S. epidermidis and S.
aureus, and more preferably, the first preparation is from S.
epidermidis (Hay, ATCC 55133).
[0031] U.S. Pat. No. 5,412,077 describes the screening of plasma
samples for effective antibody titers for the treatment or
prophylaxis of an infection caused by a respiratory virus.
[0032] Accordingly, there still remains a need to provide more
effective products and methods which make use of antibodies against
MSCRAMMs and can be utilized in methods of intravenous
immunoglobulin therapy so as to prevent and/or treat Staphylococcus
infections, and preferably those that can exhibit a broad spectrum
immunization against various strains of Staphylococcus
bacteria.
Active Immunization to Bacterial Infections
[0033] Historically, studies on bacterial adherence have focused
primarily on Gram-negative bacteria, which express a wide variety
of adhesive proteins on their cell surface (Falkow, S., et al.,
Cell, 65:1099-1102, 1992). These adhesins recognize specific
glycoconjugates exposed on the surface of host cells (particularly
epithelial layers). Employing the lectin-like structures in
attachment allows the microorganism to efficiently colonize the
epithelial surfaces. This provides the bacteria an excellent
location for replication and also the opportunity to disseminate to
neighboring host tissues. It has been demonstrated that
immunization with pilus adhesins can elicit protection against
microbial challenge, such as in Hemophilus influenza induced otitis
media in a chinchilla model (Sirakova et al., Infect. Immun,
62(5):2002-2020, 1994), Moraxella bovis in experimentally induced
infectious bovine keratoconjunctivitis (Lepper et al., Vet
Microbiol, 45(2-3):129-138, 1995), and E. coli induced diarrhea in
rabbits (McQueen et al., Vaccine, 11:201-206, 1993). In most cases,
immunization with adhesins leads to the production of immune
antibodies that prevent infection by inhibiting bacterial
attachment and colonization, as well as enhancing bacterial
opsonophagocytosis and antibody-dependent complement-mediated
killing.
[0034] The use of molecules that mediate the adhesion of pathogenic
microbes to host tissue components as vaccine components is
emerging as a critical step in the development of future vaccines.
Because bacterial adherence is the critical first step in the
development of most infections, it is an attractive target for the
development of novel vaccines. An increased understanding of the
interactions between MSCRAMMs and host tissue components at the
molecular level coupled with new techniques in recombinant DNA
technology have laid the foundation for a new generation of subunit
vaccines. Entire or specific domains of MSCRAMMs, either in their
native or site-specifically altered forms, can now be produced.
Moreover, the ability to mix and match MSCRAMMs from different
microorganisms creates the possibility of designing a single
vaccine that will protect against multiple bacteria.
[0035] The recent clinical trials with a new subunit vaccine
against whooping cough, consisting of the purified Bordatella
pertussis MSCRAMMs filamentous hemagglutinin and pertactin, in
addition to an inactivated pertussis toxin, are a prime example of
the success of this type of approach. Several versions of the new
acellular vaccine were shown to be safe and more efficacious than
the old vaccine that contained whole bacterial cells (Greco et al.,
N Eng J Med, 334:341-348, 1996; Gustaffson et al., N Eng J Med,
334:349-355, 1996).
[0036] Natural immunity to Staphylococcus infections remains poorly
understood. Typically, healthy humans and animals exhibit a high
degree of innate resistance to Staphylococcus bacteria such as S.
aureus. Protection is attributed to intact epithelial and mucosal
barriers and normal cellular and humoral responses. Titers of
antibodies to S. aureus components are elevated after severe
infections (Ryding et al., J. Med Microbiol, 43(5):328-334, 1995),
however to date there is no serological evidence of a correlation
between antibody titers and human immunity.
[0037] Over the past several decades live, heat-killed, and
formalin fixed preparations of S. aureus cells have been tested as
vaccines to prevent staphylococcal infections. A multicenter
clinical trial was designed to study the effects of a commercial
vaccine, consisting of a staphylococcus toxoid and whole killed
staphylococci, on the incidence of peritonitis, exit site
infection, and S. aureus nasal carriage among continuous peritoneal
dialysis patients (Poole-Warren, L. A., et al., Clin Nephrol,
35:198-206, 1991). Although immunization with the vaccine elicited
an increase in the level of specific antibodies to S. aureus, the
incidence of peritonitis was unaffected. Similarly, immunization of
rabbits with whole cells of S. aureus could not prevent or modify
any stage in the development of experimental endocarditis, reduce
the incidence of renal abscess, or lower the bacterial load in
infected kidneys (Greenberg, D. P., et al., Infect Immun,
55:3030-3034, 1987).
[0038] Currently there is no FDA approved vaccine for the
prevention of S. aureus infections. However, a S. aureus vaccine
(StaphVAX), based on the capsular polysaccharide, is currently
being developed by NABI (North American Biologicals Inc.). This
vaccine consists of type 5 or type 8 capsular polysaccharides
conjugated to Pseudomonas aeruginosa exotoxin A (rEPA). The vaccine
is designed to induce type-specific opsonic antibodies and enhance
opsonophagocytosis (Karakawa, W. W., et al., Infect Immun,
56:1090-1095, 1988). Using a refined lethal challenge mouse model
(Fattom, A., et al., Infect Immun, 61:1023-1032, 1996) it has been
shown that intraperitoneal infusion of type 5 specific IgG reduces
the mortality of mice inoculated intraperitoneally with S. aureus.
The type 5 capsular polysaccharide-rEPA vaccine has also been used
to vaccinate seventeen patients with end-stage renal disease
(Welch, et al., J Amer Soc Nephrol, 7(2):247-253, 1996). Geometric
mean (GM) IgG antibody levels to the type 5 conjugate increased
between 13 and 17-fold after the first immunization, however no
additional increases could be detected after additional injections.
Moreover, these vaccination regimens were not able to treat a
variety of bacterial strains.
[0039] Interestingly, the GM IgM levels of the vaccinated patients
were significantly lower than control individuals. Supported by the
animal studies, the vaccine has recently completed a Phase II trial
in continuous ambulatory peritoneal dialysis patients. The clinical
trial showed the vaccine to be safe but ineffective in preventing
staphylococcal infections (NABI SEC FORM 10-K405, 12/31/95). Two
possible explanations for the inability of StaphVAX to prevent
infections related to peritoneal dialysis in vaccinated patients
are that the immunogenicity of the vaccine was too low due to
suboptimal vaccine dosing or that antibodies in the bloodstream are
unable to affect infection in certain anatomic areas, such as the
peritoneum.
[0040] Incidence of gram-positive bacteria related sepsis is
increasing. In fact between one-third and one-half of all cases of
sepsis are caused by gram-positive bacteria, particularly S. aureus
and S. epidermidis. In the United States, it can be estimated that
over 200,000 patients will develop gram-positive related sepsis
this year. Using a mouse model (Bremell, et al., Infect Immun,
59(8):2615-2623, 1991), it has been clearly demonstrated in PCT WO
97/43314 that active immunization with M55 domain of the
Col-binding MSCRAMM can protect mice against sepsis induced death.
Mice were immunized subcutaneously with either M55 or a control
antigen (bovine serum albumin) and then challenged intravenously
with S. aureus. Eighty-three percent (35/42) of the mice immunized
with M55 survived compared to only 27% of the BSA immunized mice
(12/45). This a compilation of three separate studies.
[0041] Schennings et al. demonstrated that immunization with
fibronectin binding protein from S. aureus protects against
experimental endocarditis in rats (Micro Pathog, 15:227-236, 1993).
Rats were immunized with a fusion protein (gal-FnBP) encompassing
beta-galactosidase and the domains of fibronectin binding protein
from S. aureus responsible for binding to fibronectin. Antibodies
against gal-FnBP were shown to block the binding of S. aureus to
immobilized fibronectin in vitro. Endocarditis in immunized and
non-immunized control rats was induced by catheterization via the
right carotid artery, resulting in damaged aortic heart valves
which became covered by fibrinogen and fibronectin. The
catheterized rats were then infected intravenously with 1.times.10
5 cells of S. aureus. The number of bacteria associated with aortic
valves was determined 11/2 days after the challenge infection and a
significant difference in bacterial numbers between immunized and
non-immunized groups was then observed.
[0042] A mouse mastitis model was used by Mamo, et al., in 1994
(Vaccine, 12:988-992) to study the effect of vaccination with
fibrinogen binding proteins (especially FnBP-A) and collagen
binding protein from S. aureus against challenge infection with S.
aureus. The mice vaccinated with fibrinogen binding proteins showed
reduced rates of mastitis compared with controls. Gross examination
of challenged mammary glands of mice showed that the glands of mice
immunized with fibrinogen binding proteins developed mild
intramammary infection or had no pathological changes compared with
glands from control mice. A significantly reduced number of
bacteria could be recovered in the glands from mice immunized with
fibrinogen binding proteins as compared with controls. Mamo then
found that vaccination with FnBP-A combined with staphylococcal
alpha toxoid did not improve the protection (Mamo, et al., Vaccine,
12:988-992, 1994). Next, Mamo, et al., immunized mice with only
collagen binding protein, which did not induce protection against
the challenge infection with S. aureus.
[0043] Whole killed staphylococci were included in a vaccine study
in humans undergoing peritoneal dialysis (Poole-Warren, et al.,
Clinical Nephrology 35:198-206, 1991). In this clinical trial, a
commercially available vaccine of alpha-hemolysin toxoid combined
with a suspension of whole killed bacteria) was administered
intramuscularly ten times over 12 months, with control patients
receiving saline injections. Vaccination elicited significant
increases in the levels of antibodies to S. aureus cells in the
peritoneal fluid and to alpha-hemolysin in the serum. However,
immunization did not reduce the incidences of peritonitis,
catheter-related infections or nasal colonization among vaccine
recipients. The lack of protective efficacy in this trial was
attributed to a suboptimal vaccine formulation.
[0044] Secreted proteins have been explored as components of
subcellular vaccines. The alpha toxin is among the most potent
staphylococcal exotoxins; it has cytolytic activity, induces tissue
necrosis and kills laboratory animals. Immunization with
formaldehyde-detoxified alpha toxin does not protect animals from
systemic or localized infections, although it may reduce the
clinical severity of the infections (Ekstedt, R. D., The
Staphylococci, 385-418, 1972)
[0045] One study has evaluated the protective efficacy of
antibodies to the S. aureus microcapsule in an experimental model
of staphylococcal infection (Nemeth, J. and Lee, J. C., Infect.
Immun., 61:1023-1032, 1993). Rats were actively immunized with
killed, microencapsulated bacteria or passively immunized with
high-titer rabbit antiserum specific for the capsular
polysaccharide. Control animals were injected with saline or
passively immunized with normal rabbit serum. Protection against
catheter-induced endocarditis resulting from intravenous challenge
with the same strain was then evaluated. Despite having elevated
levels of anticapsular antibodies, the immunized animals were
susceptible to staphylococcal endocarditis and immunized and
control animals had similar numbers of bacteria in the blood.
[0046] As described in the Detailed Description of the Invention
hereinbelow, a number of patents and published patent applications
describe the gene sequences for fibronectin, fibrinogen, collagen,
elastin, and MHC II antigen type binding proteins. These patents
and patent applications are incorporated by reference in their
entirety. These documents teach that the proteins, fragments, or
antibodies immunoreactive with those proteins or fragments can be
used in vaccinations for the treatment of S. aureus infections.
PCT/US97/087210 discloses the vaccination of mice with a
combination of a collagen binding protein (M55 fragment), a
fibronectin binding peptide (formalin treated FnBPA (D1-D3)) and a
fibrinogen binding peptide (ClfA).
[0047] Despite the advances in the art of compositions for the
treatment of infections from Staphylococcus bacteria such as S.
aureus, there remains a need to provide a more effective product,
and preferably one that exhibits a broad spectrum immunization
against a variety of Staphylococcus bacterial strains. As described
in the Detailed Description of the Invention, one approach to
generating a prophylactic immunotherapeutic against bacteria is to
stimulate donors with a vaccine containing a combination of
MSCRAMMs. This approach of generating hyperimmune globulins can
create a steady supply of plasma with high levels of the specific
types of disease fighting antibodies. MSCRAMM hyperimmune globulins
can be used to provide passive immunity against infection in
neonates, trauma patients, immunocompromised patients or patients
who are immediately at risk and do not have time to mount their own
antibody response. Hyperimmune globulins have a high
benefit-to-cost ratio, can be produced from a nonhuman or human
source and have a high level of physician acceptance based on past
usage.
[0048] Therefore, it is an object of the invention to provide new
therapeutic compositions for active and passive immunization
against Staphylococcus infections.
[0049] It is another object of the present invention to provide
active and passive immunization against mastitis, arthritis,
endocarditis, septicemia, osteomyelitis, furunculosis, cellulitis,
pyemia, pneumonia, pyoderma, suppuration of wounds, food poisoning,
bladder infections and other infectious diseases.
[0050] It is another object of the present invention to provide a
therapeutic composition that immunizes against Staphylococcus
bacteria such as S. aureus and S. epidermidis, increases the rate
of opsonization and phagocytosis of a variety of Staphylococcus
infections, and induces enhanced intracellular killing of
Staphylococcus bacteria.
[0051] It is another object of the present invention to provide an
immunological serum against staphylococci.
[0052] It is another object of the present invention to provide
such a serum which yields humoral and cellular immunity against
staphylococci.
[0053] It is another object of the present invention to provide
such a serum which imparts short-term immunity against
staphylococci.
[0054] It is a further object of the present invention to provide
methods for detecting, diagnosing, treating, preventing or
monitoring the progress of therapy for staphylococcal
infections.
SUMMARY OF THE INVENTION
[0055] A method and composition for the passive immunization of
patients infected with or susceptible to infection from
Staphylococcus bacteria such as S. aureus and S. epidermidis
infection is provided that includes the selection or preparation of
a donor plasma pool with high antibody titers to carefully selected
Staphylococcus adhesins or MSCRAMMs, or fragments or components
thereof, or sequences with substantial homology thereto;
purification, concentration, and treatment of the donor plasma pool
as necessary to obtain immunoglobulin in a purified state that has
a higher than normal antibody titer to the selected adhesins; and
then administration of an effective amount of the purified
immunoglobulin to the patient in need thereof. The donor plasma
pool can be prepared, for example, by combining individual blood or
blood component samples which have higher than normal titers of
antibodies to one or more of the selected adhesins or other
proteins that bind to extracellular matrix proteins, or fragments
or sequences with substantial homology thereto, to produce the
desired composite. Kits for the identification of donor plasma
pools with high titers of the selected adhesins are also provided.
In an alternative embodiment, a method for obtaining a donor plasma
pool that is highly effective against Staphylococcus bacterial
infection is provided that includes administering carefully
selected proteins or peptides to a host to induce the expression of
desired antibodies, recovering the enhanced high titer serum or
plasma pool from the host, optionally purifying and concentrating
the immunoglobulin, and providing it to a patient in need
thereof.
[0056] A "high titer" of antibody in this context means the
presence of an antibody which is immunoreactive with the selected
adhesin or fragment thereof which is 2-fold or greater, e.g., up to
10-20 more times higher than that found in a normal population of
100 random samples of blood or blood components.
[0057] In one embodiment of the invention, a donor plasma
composition is selected or prepared that has a high titer of
antibodies to at least a fibrinogen binding protein, such as
Clumping factor A ("ClfA") or Clumping factor B ("ClfB"), or
fragments or components thereof, or a protein or fragment with
sufficiently high homology thereto.
[0058] In another embodiment of the invention, a donor plasma
composition is selected or prepared that has a high titer of
antibodies to at least a collagen binding protein or peptide (or an
appropriate site directed mutated sequence thereof), a fragment or
component thereof, such as the collagen binding domain protein M55,
or a protein or fragment with sufficiently high homology
thereto.
[0059] In another embodiment of the invention, a donor plasma
composition is selected or prepared that has a high titer of
antibodies to at least a fibronectin binding protein or peptide (or
an appropriate site directed mutated sequence thereof), or a
protein or fragment with sufficiently high homology thereto, as
well as the fibrinogen binding protein A and B (ClfA or ClfB), or
useful fragments or components thereof or a protein or fragment
with sufficiently high homology thereto.
[0060] In a further embodiment, a donor pool is selected or
prepared that has a high titer of antibodies to at least the
fibrinogen binding protein A (ClfA) and the fibrinogen binding
protein B (ClfB), or useful fragments thereof or a protein or
fragment with sufficiently high homology thereto.
[0061] In a still further embodiment, a donor pool is selected or
prepared with a high titer of antibodies to at least a fibronectin
binding protein or peptide (or an appropriate site directed mutated
sequence thereof), or a protein or fragment with sufficiently high
homology thereto, in combination with (I) high titer antibodies to
the fibrinogen binding protein A and B (ClfA and ClfB), or a useful
fragment thereof or a protein or fragment with sufficiently high
homology thereto; and (ii) high titer antibodies to a collagen
binding protein or useful fragment thereof.
[0062] In another embodiment, a donor pool is selected or prepared
that has a high titer of antibodies as in any of the previous
embodiments in combination with a high titer of antibodies to an
elastin binding protein or peptide or a protein or fragment with
sufficiently high homology thereto.
[0063] In another embodiment, a donor pool is selected or prepared
that has a high titer of antibodies as set forth in the embodiments
above in combination with high titers of antibodies to a MHC II
analogous protein or peptide or a protein or fragment with
sufficiently high homology thereto.
[0064] In an additional embodiment, a donor pool is selected or
prepared that has a high titer of antibodies to any of the
embodiments above in combination with high titer of antibodies to
one or more fibrinogen binding proteins SdrC, SdrD or SdrE, or
useful fragments thereof or proteins or fragments with sufficiently
high homology thereto.
[0065] In still another embodiment, a donor pool is selected or
prepared that has a high titer of antibodies to at least the
fibrinogen binding protein SdrC, the fibrinogen binding protein
SdrD and the fibrinogen binding protein SdrE or useful fragments
thereof or a protein or fragment with sufficiently high homology
thereto.
[0066] Kits are also provided that identify plasma pools with high
titers of the desired antibodies. In one embodiment, a suitable
amount of antibodies to antibodies of the combination of proteins
or peptides as described herein can be immobilized on a solid
support and are preferably labeled with a detectable agent.
Antibodies can be immobilized to a variety of solid substrates by
known methods. Suitable solid support substrates include materials
having a membrane or coating supported by or attached to sticks,
beads, cups, flat packs, or other solid support. Other solid
substrates include cell culture plates, ELISA plates, tubes, and
polymeric membranes. The antibodies can be labeled with a
detectable agent such as a fluorochrome, a radioactive label,
biotin, or another enzyme, such as horseradish peroxidase, alkaline
phosphatase and 2-galactosidase. If the detectable agent is an
enzyme, a means for detecting the detectable agent can be supplied
with the kit. A preferred means for detecting a detectable agent
employs an enzyme as a detectable agent and an enzyme substrate
that changes color upon contact with the enzyme. The kit can also
contain a means to evaluate the product of the assay, for example,
a color chart, or numerical reference chart.
[0067] Preferably, the isolated immunoglobulin is of the IgG
fraction or isotype, but isolated immunoglobulin is not restricted
to any particular fraction or isotype and may be IgG, IgM, IgA,
IgD, IgE, or any combination thereof. It is also preferable that
the isolated immunoglobulin be purely or antigenically human
immunoglobulin, which may be obtained from human sources or made
directly by the fusion of human antibody producing cells with human
antibody producing cells or by the substitution of human DNA
sequences for some of the nonhuman DNA sequences which code for the
antibody while retaining the antigen binding ability of the
original antibody molecule.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0068] FIG. 1 is a schematic representation of the peptides used in
illustrative vaccine, MSCRAMM IV. This drawing illustrates the
essential features of the collagen binding MSCRAMM CNA, fibrinogen
binding MSCRAMM ClfA, fibrinogen binding MSCRAMM ClfB and
fibronectin binding MSCRAMM FnBPA proteins.
[0069] FIG. 2 is a time course graph of the immune response in
MCSCRAMM vaccinated Rhesus Monkeys as shown by changes in antibody
titers against the MSCRAMMs CNA, ClfA, ClfB and FnBPA,
respectively. The titers were analyzed by ELISA and measured as
changes in absorbance (quantified at 405 nm) during each week over
the course of a six-month period of treatment following the
original immunization with the antigen.
DETAILED DESCRIPTION OF THE INVENTION
[0070] A method and composition for the passive immunization of
patients infected with or susceptible to Staphylococcus bacterial
infection, such as those caused by S. aureus or S. epidermidis, is
provided that includes the selection or preparation of a donor
plasma pool with high antibody titers to carefully selected
Staphylococcus adhesins, or fragments thereof or sequences with
substantial homology thereto; purification, concentration, and
treatment of the donor plasma pool as necessary to obtain
immunoglobulin in a purified state that has a higher than normal
antibody titer to the selected staphylococcal adhesins; and then
administration of an effective amount of the purified
immunoglobulin to the patient in need thereof. The donor plasma
pool can be prepared, for example by, by combining individual blood
samples which have higher than normal titers of antibodies to one
or more of the selected adhesins or fragments or sequences with
substantial homology thereto. Kits for the identification of donor
plasma pools with high titers of the selected adhesins are also
provided. In an alternative embodiment, a method for obtaining a
donor plasma pool that is highly effective against Staphylococcus
infection is provided that includes administering carefully
selected proteins or peptides to a host to induce the expression of
desired antibodies, recovering the enhanced high titer plasma pool
from the host, optionally purifying and concentrating the
immunoglobulin, and providing it to a patient in need thereof.
[0071] Donor plasma pools are selected or prepared, purified,
treated, and then administered in an effective amount to a patient
in need thereof, which include high titer antibodies to at
least:
[0072] (i) a fibrinogen binding protein, such as Clumping factor A
("ClfA") or Clumping factor B ("ClfB"), or fragments or components
thereof, or a protein or fragment with sufficiently high homology
thereto;
[0073] (ii) a collagen binding protein or peptide (or an
appropriate site directed mutated sequence thereof), a fragment or
component thereof, such as the collagen binding domain protein M55,
or a protein or fragment with sufficiently high homology
thereto.
[0074] (iii) a fibronectin binding protein or peptide (or an
appropriate site directed mutated sequence thereof), or a protein
or fragment with sufficiently high homology thereto, in combination
with the fibrinogen binding protein A and B (ClfA and ClfB), or
useful fragments thereof or a protein or fragment with sufficiently
high homology thereto;
[0075] (iv) the fibrinogen binding protein A (ClfA) and the
fibrinogen binding protein B (ClfB), or useful fragments thereof or
a protein or fragment with sufficiently high homology thereto;
[0076] (v) fibronectin binding protein or peptide (or an
appropriate site directed mutated sequence thereof), or a protein
or fragment with sufficiently high homology thereto, in combination
with (I) the fibrinogen binding protein A and B (ClfA and ClfB), or
a useful fragment thereof or a protein or fragment with
sufficiently high homology thereto; and (ii) a collagen binding
protein or useful fragment thereof;
[0077] (vi) components of any of the above in combination with an
elastin binding protein or peptide or a protein or fragment with
sufficiently high homology thereto;
[0078] (vii) components of any of the above embodiments in
combination with a MHC II type binding protein or peptide or a
protein or fragment with sufficiently high homology thereto;
[0079] (viii) components of any of the above embodiments in
combination with a the fibrinogen binding proteins SdrC, SdrD or
SdrE, or useful fragments thereof or proteins or fragments with
sufficiently high homology thereto;
[0080] (ix) the fibrinogen binding protein SdrC, the fibrinogen
binding protein SdrD and the fibrinogen binding protein SdrE or
useful fragments thereof or a protein or fragment with sufficiently
high homology thereto; or
[0081] (x) proteins SdrF, SdrG and SdrH from coagulase-negative
bacteria such as S. epidermidis or useful fragments thereof or a
proteins or fragments with sufficiently high homology thereto.
[0082] Isolated peptide fragments from wild-type or naturally
occurring variants and synthetic or recombinant peptides
corresponding to wild-type, naturally occurring variants or
introduced mutations that do not correspond to a naturally
occurring binding domain of a binding protein can be used to select
or produce donor plasma pools.
I. Definitions
[0083] The terms FnBP-A protein, FnBP-B protein, ClfA protein, ClfB
protein, SdrC protein, SdrD protein, SdrE protein, CNA protein,
EbpS protein and MHCII protein are defined herein to include
FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHCII
subdomains, respectively, and active or antigenic fragments or
components of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA,
EbpS and MHCII proteins, respectively, or proteins or fragments
having sufficiently high homology thereto. Active fragments or
components of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA,
EbpS and MHCII proteins are defined herein as peptides or
polypeptides capable of blocking the binding of staphylococci
bacteria to extracellular matrix proteins of the host. Antigenic
fragments of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA,
EbpS and MHCII proteins are defined herein as peptides or
polypeptides capable of producing an immunological response.
[0084] The term "adhesin" as used herein includes naturally
occurring and synthetic or recombinant proteins and peptides which
can bind to extracellular matrix proteins and/or mediate adherence
to host cells.
[0085] The term "amino acid" as used herein includes naturally
occurring and synthetic amino acids and includes, but is not
limited to, alanine, valine, leucine, isoleucine, proline,
phenylalanine, tryptophan, methionine, glycine, serine, threonine,
cysteine, tyrosine, asparagine, glutamate, aspartic acid, glutamic
acid, lysine, arginine, and histidine.
[0086] An "antibody" is any immunoglobulin, including antibodies
and fragments thereof, that binds a specific epitope. The term as
used herein includes monoclonal antibodies, polyclonal, chimeric,
single chain, bispecific, simianized, and humanized-antibodies as
well as Fab fragments, including the products of an Fab
immunoglobulin expression library.
[0087] The phrase "antibody molecule" in its various grammatical
forms as used herein contemplates both an intact immunoglobulin
molecule and an immunologically active portion of an immunoglobulin
molecule.
[0088] As used herein, "pg" means picogram, "ng" means nanogram,
"ug" or ".mu.g" mean microgram, "mg" means milligram, "ul" or
".mu.l" mean microliter, "ml" means milliliter, "l" means
liter.
[0089] A "cell line" is a clone of a primary cell that is capable
of stable growth in vitro for many generations.
[0090] A "clone" is a population of cells derived from a single
cell or common ancestor by mitosis.
[0091] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the sequence are determined by a start codon at the
5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genetic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence.
[0092] "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
its either single stranded form, or a double stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g, restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA
[0093] As used herein, the term "extracellular matrix proteins," or
ECM, refers to four general families of macromolecules--collagens,
structural glycoproteins, proteoglycans and elastins--that provide
support and modulate cellular behavior.
[0094] "Immunologically effective amounts" are those amounts
capable of stimulating a B cell and/or T cell response.
[0095] As used herein, the term "in vivo vaccine" refers to
immunization of animals with proteins so as to elicit a humoral and
cellular response that protects against later exposure to the
pathogen.
[0096] The term "ligand" is used to include molecules, including
those within host tissues, to which pathogenic bacteria attach.
[0097] The term "MHC II analogous proteins" as used herein refers
to cell-surface molecules that are responsible for rapid graft
rejections and are required for antigen presentation to
T-cells.
[0098] The phrase "monoclonal antibody" in its various grammatical
forms refers to an antibody having only one species of antibody
combining site capable of immunoreacting with a particular
antigen.
[0099] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are
physiologically tolerable and do not typically produce an
unacceptable allergic or similar untoward reaction when
administered to a human.
[0100] As used herein, a "protective antibody" is an antibody which
confers protection against infectious diseases caused by infection
with staphylococci, when used to passively immunize an naive
animal.
[0101] As used herein, a "protective epitope" is an epitope which
is recognized by a protective antibody, and/or an epitope which,
when used to immunize an animal, elicits an immune response
sufficient to prevent or lessens the severity for some period of
time, of any one of the disorders which can result from infection
with staphylococci.
[0102] The term "wound" is used herein to mean that normally
covering epithelial cellular layer, and other surface structures
have been damaged by mechanical, chemical or other influence.
[0103] By "immunologically effective amount" is meant an amount of
a peptide composition that is capable of generating an immune
response in the recipient animal. This includes both the generation
of an antibody response (B cell response), and/or the stimulation
of a cytotoxic immune response (T cell response). The generation of
such an immune response will have utility in both the production of
useful bioreagents, e.g., CTLs and, more particularly, reactive
antibodies, for use in diagnostic embodiments, and will also have
utility in various prophylactic or therapeutic embodiments.
II. Fibronectin-Binding MSCRAMMs
[0104] Fibronectin (Fn) is a 440-kDa glycoprotein found in the ECM
and body fluids of animals. The primary biological function of
fibronectin appears to be related to its ability to serve as a
substrate for the adhesion of cells expressing the appropriate
integrins. Several bacterial species have been shown to bind
fibronectin specifically and to adhere to a fibronectin-containing
substratum. Most S. aureus isolates bind Fn, but do so in varying
extents, which reflects variations in the number of MSCRAMM
molecules expressed on the bacterial cell surface. The interaction
between Fn and S. aureus is highly specific (Kuusela, P., Nature,
276:718-20, 1978). Fn binding is mediated by two surface exposed
proteins with molecular weights of 110 kDa, named FnBP-A and
FnBP-B. The primary Fn binding site consists of a motif of 35-40
amino acids, repeated three to five times. The genes for these have
been cloned and sequenced (Jonsson, K., et al., Eur. J. Biochem.,
202:1041-1048, 1991). Potential applications for vaccination with
anti-FnBP antibodies include, but are not limited to, bovine
mastitis, endocarditis and wound infections.
[0105] WO-A-85/05553 discloses bacterial cell surface proteins
having fibronectin, fibrinogen, collagen, and or laminin binding
ability.
[0106] U.S. Pat. Nos. 5,320,951 and 5,571,514 to Hook, et al.,
discloses the gene sequence of fibronectin binding protein A
(fnbA), and biological products and methods based on this
sequence.
[0107] U.S. Pat. No. 5,175,096 to Hook et al., discloses the gene
sequence of fnbB, a hybrid DNA molecule (fnbB) and biological
products and methods based on this sequence.
[0108] U.S. Pat. No. 5,652,217 discloses an isolated and purified
protein having binding activity that is encoded by a hybrid DNA
molecule from S. aureus of defined sequence.
[0109] U.S. Pat. No. 5,440,014 discloses a fibronectin binding
peptide within the D3 homology unit of a fibronectin binding
protein of S. aureus which can be used for vaccination of ruminants
against mastitis caused by staphylococcal infections, for treatment
of wounds, for blocking protein receptors, for immunization of
other animals, or for use in a diagnostic assay.
[0110] U.S. Pat. No. 5,189,015 discloses a method for the
prophylactic treatment of the colonization of a S. aureus bacterial
strain having the ability to bind to fibronectin in a mammal that
includes administering to the mammal in need of treatment a
prophylactically therapeutically active amount of a protein having
fibronectin binding properties, to prevent the generation of
infections caused by a S. aureus bacterial strain having the
ability to bind fibronectin, wherein the protein has a molecular
weight of 87 kDa to 165 kDa.
[0111] U.S. Pat. No. 5,416,021 discloses a fibronectin binding
protein encoding DNA from Streptococcus dysgalactiae, along with a
plasmid that includes DNA encoding for fibronectin binding protein
from S. dysgalactiae contained in E. coli, DNA encoding a
fibronectin binding protein from S. dysgalactiae and an E. coli
microorganism transformed by DNA encoding a fibronectin binding
protein from S. dysgalactiae.
[0112] It has been observed that antibodies to wild type
fibronectin binding protein do not substantially inhibit the
ability of S. aureus to bind to fibronectin, and thus do not
exhibit a significant therapeutic effect in vivo. PCT/US98/01222
discloses antibodies that block the binding of fibronectin to
fibronectin binding proteins. The antibodies were raised against a
site-directed mutated sequence of fibronectin binding protein that
does not bind to fibronectin. It was identified that there is a
rapid complexing of fibronectin with fibronectin binding proteins
and fragments in vivo. Peptide epitopes that do not bind to
fibronectin, even though based on a fibronectin binding domain of a
fibronectin binding protein, do not form a complex with fibronectin
in vivo. This allows antibodies to be made against the uncomplexed
peptide epitope, which inhibit or block the binding of fibronectin
to fibronectin binding proteins.
III. Collagen-Binding MSCRAMMs
[0113] Collagen is the major constituent of cartilage. Collagen
(Cn) binding proteins are commonly expressed by staphylococcal
strains. The Cn binding MSCRAMM of S. aureus adheres to cartilage
in a process that constitutes an important part of the pathogenic
mechanism in staphylococcal infections. (Switalski, et al. Mol.
Micro. 7(1), 99-107, 1993) Cn binding by staphylococcal bacteria
such as S. aureus is found to play a role in at least, but not
only, arthritis and septicemia. CNA proteins with molecular weights
of 133, 110 and 87 kDa (Patti, J., et al., J. Biol. Chem.,
267:4766-4772, 1992) have been identified. Strains expressing CNAs
with different molecular weights do not differ in their Cn binding
ability (Switalski, L. M., et al., Mol. Microbiol., 7:99-107,
1993).
[0114] Staphylococcal strains recovered from the joints of patients
diagnosed with septic arthritis or osteomyelitis almost invariably
express a CNA, whereas significantly fewer isolates obtained from
wound infections express this adhesin. (Switalski, L. M., et al.,
Mol. Microbiol., 7:99-107, 1993) Similarly, S. aureus strains
isolated from the bones of patients with osteomyelitis more often
have an MSCRAMM recognizing the bone-specific protein, bone
sialoprotein (BSP) (Ryden, C., et al, Lancet, 11:515-518, 1987). S.
aureus colonization of the articular cartilage within the joint
space appears to be an important factor contributing to the
development of septic arthritis.
[0115] The cloning, sequencing, and expression of a gene CNA,
encoding a S. aureus CNA protein has been reported (Patti, J., et
al., J. Biol. Chem., 267:4766-4772, 1992). The CNA gene encodes an
133-kDa adhesin that contains structural features characteristic of
surface proteins isolated from Gram-positive bacteria.
[0116] Recently, the ligand-binding site has been localized within
the N-terminal half of the CNA (Patti, J. et al., Biochemistry,
32:11428-11435, 1993). By analyzing the Col binding activity of
recombinant proteins corresponding to different segments of the
MSCRAMM, a 168-amino-acid long protein fragment (corresponding to
amino acid residues 151-318) that had appreciable Col binding
activity was identified. Short truncations of this protein in the N
or C terminus resulted in a loss of ligand binding activity but
also resulted in conformational changes in the protein.
[0117] PCT WO 92/07002 discloses a hybrid DNA molecule which
includes a nucleotide sequence from S. aureus coding for a protein
or polypeptide having collagen binding activity and a plasmid or
phage comprising the nucleotide sequence. Also disclosed are an E.
coli strain expressing the collagen binding protein, a
microorganism transformed by the recombinant DNA, the method for
producing a collagen binding protein or polypeptide, and the
protein sequence of the collagen binding protein or
polypeptide.
[0118] Patti et al. (J of Biol Chem., 270, 12005-12011, 1995)
disclose a collagen binding epitope in the S. aureus adhesin
encoded by the CNA gene. In this study, the authors synthesized
peptides derived from the sequence of the said protein and used
them to produce antibodies. Some of these antibodies inhibit the
binding of the protein to collagen.
[0119] PCT/US97/08210 discloses that certain identified epitopes of
the collagen binding protein (M55, M33, and M17) can be used to
generate protective antibodies. The application also discloses the
crystal structure of the CNA which provides critical information
necessary for identifying compositions which interfere with, or
block completely, the binding of Col to CNAs. The ligand-binding
site in the S. aureus CNA and a 25-amino-acid peptide was
characterized that directly inhibits the binding of S. aureus to
125 I-labeled type II Col.
IV. Fibrinogen-Binding MSCRAMMs
[0120] Fibrin is the major component of blood clots, and
fibrinogen/fibrin is one of the major host proteins deposited on
implanted biomaterials. Considerable evidence exists to suggest
that bacterial adherence to fibrinogen/fibrin is important in the
initiation of device-related infection. For example, as shown by
Vaudaux et al., S. aureus adheres to in vitro plastic that has been
coated with fibrinogen in a dose-dependent manner (J. Infect. Dis.
160:865-875 (1989)). In addition, in a model that mimics a blood
clot or damage to a heart valve, Herrmann et al. demonstrated that
S. aureus binds avidly via a fibrinogen bridge to platelets
adhering to surfaces (J. Infect. Dis. 167: 312-322 (1993)). S.
aureus can adhere directly to fibrinogen in blood clots formed in
vitro, and can adhere to cultured endothelial cells via fibrinogen
deposited from plasma acting as a bridge (Moreillon et al., Infect.
Immun. 63:4738-4743 (1995); Cheung et al., J. Clin. Invest.
87:2236-2245 (1991)). As shown by Vaudaux et al. and Moreillon et
al., mutants defective in the fibrinogen-binding protein clumping
factor (ClfA) exhibit reduced adherence to fibrinogen in vitro, to
explanted catheters, to blood clots, and to damaged heart valves in
the rat model for endocarditis (Vaudaux et al., Infect. Immun.
63:585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-4743
(1995)).
[0121] An adhesin for fibrinogen, often referred to as "clumping
factor," is located on the surface of S. aureus cells. The
interaction between bacteria and fibrinogen in solution results in
the instantaneous clumping of bacterial cells. The binding site on
fibrinogen is located in the C-terminus of the gamma chain of the
dimeric fibrinogen glycoprotein. The affinity is very high and
clumping occurs in low concentrations of fibrinogen. Scientists
have recently shown that clumping factor also promotes adherence to
solid phase fibrinogen, to blood clots, and to damaged heart valves
(McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); Vaudaux et
al., Infect. Immun. 63:585-590 (1995); Moreillon et al., Infect.
Immun. 63: 4738-4743 (1995)).
[0122] Two genes in S. aureus have been found that code for two Fg
binding proteins, ClfA and ClfB. The gene, clfA, was cloned and
sequenced and found to code for a polypeptide of 92 kDa. ClfA binds
the gamma chain of fibronectin, and ClfB binds the alpha and beta
chains (Eidhin, et al., Mol Micro, awaiting publication, 1998).
ClfB is a cell wall associated protein with a predicted molecular
weight of 88 kDa and an apparent molecular weight of 124 kDa that
binds both soluble and immobilized fibrinogen and acts as a
clumping factor.
[0123] The gene for a clumping factor protein, designated ClfA, has
recently been cloned, sequenced and analyzed in detail at the
molecular level (McDevitt et al., Mol. Microbiol. 11: 237-248
(1994); McDevitt et al., Mol. Microbiol. 16:895-907 (1995)). The
predicted protein is composed of 933 amino acids. A signal sequence
of 39 residues occurs at the N-terminus followed by a 520 residue
region (region A), which contains the fibrinogen binding domain. A
308 residue region (region R), composed of 154 repeats of the
dipeptide serine-aspartate, follows. The R region sequence is
encoded by the 18 basepair repeat GAY TCN GAY TCN GAY AGY in which
Y equals pyrimidines and N equals any base. The C-terminus of ClfA
has features present in many surface proteins of gram-positive
bacteria such as an LPDTG motif, which is responsible for anchoring
the protein to the cell wall, a membrane anchor, and positive
charged residues at the extreme C-terminus.
[0124] The platelet integrin alpha IIb.beta.3 recognizes the
C-terminus of the gamma chain of fibrinogen. This is a crucial
event in the initiation of blood clotting during coagulation. ClfA
and alpha IIb.beta.3 appear to recognize precisely the same sites
on fibrinogen gamma chain because ClfA can block platelet
aggregation, and a peptide corresponding to the C-terminus of the
gamma chain (198-41 1) can block both the integrin and ClfA
interacting with fibrinogen (McDevitt et al., Eur. J. Biochem.
247:416-424 (1997)). The fibrinogen binding site of alpha
IIb.beta.33 is close to, or overlaps, a Ca2+ binding determinant
referred to as an "EF hand". ClfA region A carries several EF
hand-like motifs. A concentration of Ca2+ in the range of 3-5 mM
blocks these ClfA-fibrinogen interactions and changes the secondary
structure of the ClfA protein. Mutations affecting the ClfA EF hand
reduce or prevent interactions with fibrinogen. Ca2+ and the
fibrinogen gamma chain seem to bind to the same, or to overlapping,
sites in ClfA region A.
[0125] The alpha chain of the leukocyte integrin, alpha MB2, has an
insertion of 200 amino acids (A or I domain) which is responsible
for ligand binding activities. A novel metal ion-dependent adhesion
site (MIDAS) motif in the I domain is required for ligand binding.
Among the ligands recognized is fibrinogen. The binding site on
fibrinogen is in the gamma chain (residues 190-202). It was
recently reported that Candida albicans has a surface protein,
alpha Intip, having properties reminiscent of eukaryotic integrins.
The surface protein has amino acid sequence homology with the 1
domain of M.beta.2, including the MIDAS motif. Furthermore, Intlp
binds to fibrinogen.
[0126] ClfA region A also exhibits some degree of sequence homology
with alpha Intlp. Examination of the ClfA region A sequence has
revealed a potential MIDAS motif. Mutations in putative cation
coordinating residues in the D.times.S.times.S portion of the MIDAS
motif in ClfA results in a significant reduction in fibrinogen
binding. A peptide corresponding to the gamma-chain binding site
for alpha M.beta.2 (190-202) has been shown by O'Connell et al. to
inhibit ClfA-fibrinogen interactions (O'Connell et al., J. Biol.
Chem., in press). Thus it appears that ClfA can bind to the
gamma-chain of fibrinogen at two separate sites. The ligand binding
sites on ClfA are similar to those employed by eukaryotic integrins
and involve divalent cation binding EF-hand and MIDAS motifs.
Despite the low level of identity between ClfA and ClfB, both
proteins bind fibrinogen (on different chains) by a mechanism that
is susceptible to inhibition by divalent cations, despite not
sharing obvious metal binding motifs.
[0127] Other fibrinogen binding proteins are disclosed in
co-pending U.S. patent application Ser. No. 09/200,650,
incorporated herein by reference. This application discloses
isolated fibrinogen binding proteins ClfB, SdrC, SdrD and SdrE as
well as antibodies to the proteins and diagnostic kits that include
the proteins or the antibodies. Also claimed are a method of
preventing a S. aureus infection that includes administering to the
patient an effective amount of ClfB, SdrC, SdrD, SdrE, or a binding
fragment thereof and a method of inducing an immunological response
comprising administering to a patient a pharmaceutical composition
that includes ClfB, SdrC, SdrD, SdrE, or an active fragment
thereof.
[0128] ClfB has a predicted molecular weight of approximately 88
kDa and an apparent molecular weight of approximately 124 kDa. ClfB
is a cell-wall associated protein and binds both soluble and
immobilized fibrinogen. In addition, ClfB binds both the alpha and
beta chains of fibrinogen and acts as a clumping factor. The ClfB
protein has been demonstrated to be a virulence factor in
experimental endocarditis.
[0129] The SdrC, SdrD and SdrE proteins are related in primary
sequence and structural organization to the ClfA and ClfB proteins
and are localized on the cell surface. The SdrC, SdrD and SdrE
proteins are cell wall-associated proteins, having a signal
sequence at the N-terminus and an LPXTG (SEQ ID NO: 2) motif,
hydrophobic domain and positively charged residues at the
C-terminus. Each also has an SD repeat containing region R of
sufficient length to allow efficient expression of the ligand
binding domain region A on the cell surface. With the A region of
the SdrC, SdrD and SdrE proteins located on the cell surface, the
proteins can interact with proteins in plasma, the extracellular
matrix or with molecules on the surface of host cells. They share
some limited amino acid sequence similarity with ClfA and ClfB.
Additionally, SdrC, SdrD and SdrE also exhibit cation-dependent
ligand binding to extracellular matrix proteins. For example, SdrC
binds vitronectin and SrdE binds bone sialoprotein (BSP).
[0130] It has been discovered that in the A region of SrdC, SrdD,
SrdE, ClfA and ClfB there is a highly conserved amino acid sequence
that can be used to derive a consensus TYTFTDYVD (SEQ ID NO: 3)
motif. The motif can be used in multicomponent vaccines to impart
broad spectrum immunity to bacterial infections, and also can be
used to produce monoclonal or polyclonal antibodies that impart
broad spectrum passive immunity. In an alternative embodiment, any
combination of the variable sequence motif derived from the Sdr and
Clf protein families, (T/l) (Y/F) (TN) (F) (T) (D/N) (Y) (V) (D/N),
can be used to impart immunity or produce protective
antibodies.
[0131] ClfB, SdrC, SdrD and SdrE thus share a common consensus
TYTFTDYVD (SEQ ID NO: 3) motif which overlaps the ligand
binding/Ca2+ binding region of ClfA. Therefore the proteins
interact with fibrinogen and other host components. ClfB, SdrC,
SdrD and SdrE subdomains, depending on the protein, include
subdomains A and B1-B5. Other information regarding extracellular
matrix binding proteins has been disclosed in U.S. application Ser.
No. 09/200,650, incorporated herein by reference.
V. Elastin-Binding MSCRAMMs
[0132] The primary role of elastin is to confer the property of
reversible elasticity to tissues and organs (Rosenbloom, J., et
al., FASEB J., 7:1208-1218, 1993). Elastin expression is highest in
the lung, skin and blood vessels, but the protein is widely
expressed in mammalian hosts for S. aureus. S. aureus binding to
elastin was found to be rapid, reversible, of high affinity and
ligand specific. Furthermore, a 25 kDa cell surface elastin binding
protein (EbpS) was isolated and proposed to mediate S. aureus
binding to elastin-rich host ECM. EbpS binds to a region in the
N-terminal 30 kDa fragment of elastin.
[0133] PCT/US97/03106 discloses the gene sequences for an elastin
binding protein. DNA sequence data disclosed indicates that the
ebps open reading frame consists of 606 bp, and encodes a novel
polypeptide of 202 amino acids. EbpS protein has a predicted
molecular mass of 23,345 daltons and pI of 4.9. EbpS was expressed
in E. coli as a fusion protein with polyhistidine residues attached
to the N-terminus. A polyclonal antibody raised against recombinant
EbpS interacted specifically with the 25 kDa cell surface EbpS and
inhibited staphylococcal elastin binding. Furthermore, recombinant
EbpS bound specifically to immobilized elastin and inhibited
binding of Staphylococcus aureus to elastin. A degradation product
of recombinant EbpS lacking the first 59 amino acids of the
molecule and a C-terminal fragment of CNBr-cleaved recombinant
EbpS, however, did not interact with elastin. These results
strongly suggest that EbpS is the cell surface molecule mediating
binding of Staphylococcus aureus to elastin. The finding that some
constructs of recombinant EbpS do not interact with elastin
suggests that the elastin binding site in EbpS is contained in the
first 59 amino acids of the molecule.
[0134] Several independent criteria indicate that EbpS is the
surface protein mediating cellular elastin binding. First, rEbpS
binds specifically to immobilized elastin and inhibits binding of
S. aureus cells to elastin in a dose dependent manner. These
results establish that EbpS is an elastin binding protein that is
functionally active in a soluble form. Second, an antibody raised
against rEbpS recognizes a 25 kDa protein expressed on the cell
surface of S. aureus cells. In addition to the size similarity and
antibody reactivity, further evidence that this 25 kDa protein is
cell surface EbpS is provided by the experiment showing that
binding of the 25 kDa protein to immobilized anti-rEbpS IgG is
inhibited in the presence of excess unlabeled rEbpS. Finally, Fab
fragments prepared from the anti-rEbpS antibody, but not from its
pre-immune control, inhibit binding of S. aureus to elastin. This
result suggests that the topology of surface EbpS is such that the
elastin binding site is accessible to interact with ligands (i.e.
elastin and the anti-rEbpS Fab fragment) and not embedded in the
cell wall or membrane domains. The composite data demonstrate that
EbpS is the cell surface protein responsible for binding S. aureus
to elastin.
[0135] The present and previous findings suggest the existence of a
functionally active 40 kDa intracellular precursor form of EbpS
that requires processing at the C-terminus prior to surface
expression. This notion is based on the following observations: i)
there exists an intracellular 40 kDa elastin binding protein that
is never detected during cell surface labeling experiments, ii) the
25 kDa EbpS and the 40 kDa elastin binding protein have an
identical N-terminal sequence, and iii) a single gene exists for
EbpS. Because the size of the ebps open reading frame is not
sufficient to encode a 40 kDa protein, at first the inventors
disregarded this hypothesis. However, their studies with rEbpS
demonstrated that although the actual size of the recombinant
protein is 26 kDa, it migrates aberrantly as a 45 kDa protein in
SDS-30 PAGE. This finding suggests that full length native EbpS,
with a predicted size of 23 kDa, may be migrating in SDS-PAGE as
the 40 kDa intracellular precursor, and that the 25 kDa surface
form of EbpS is actually a smaller form of the molecule processed
at the C-terminus. Although EbpS lacks an N-terminal signal peptide
and other known sorting and anchoring signals, this proposed
intracellular processing event may explain some questions regarding
how EbpS is targeted to the cell surface. In fact, C-terminal
signal peptides have been identified in several bacterial proteins
(Fath, M. J. and Kolter, R., Microbiol. Rev., 57:995-1017, 1993)
and alternative means of anchoring proteins to the cells surface
have been reported in gram positive bacteria (Yother, J. and White,
J. M., J. Bacteriol., 176:2976-2985, 1994).
[0136] Using overlapping EbpS fragments and recombinant constructs,
the elastin binding site in EbpS was mapped to the amino terminal
domain of the molecule (PCT/US97/03106). Overlapping synthetic
peptides spanning amino acids 14-34 were then used to better define
the binding domain. Among these, peptides corresponding to residues
14-23 and 18-34 specifically inhibited elastin binding by more than
95%. Common to all active synthetic peptides and proteolytic and
recombinant fragments of EbpS is the hexameric sequence
.sup.18Thr-Asn-Ser-His-Gln-Asp.sup.23. Further evidence that this
sequence is important for elastin binding was the loss of activity
when Asp.sup.23 was substituted with Asn in the synthetic peptide
corresponding to residues 18-34. However, the synthetic hexamer
TNSHQD by itself did not inhibit staphylococcal binding to elastin.
These findings indicate that although the presence of the TNSHQD
sequence is essential for EbpS activity, flanking amino acids in
the N- or C-terminal direction and the carboxyl side chain of
Asp.sup.23 are required for elastin recognition.
VI. MHC II--Analogous Proteins, (MAP)
[0137] In addition to fibrinogen, fibronectin, collagen and
elastin, S. aureus strains associate with other adhesive eukaryotic
proteins, many of which belong to the family of adhesive matrix
proteins, such as vitronectin. (Chatwal, G. S., et al., Infect.
Immun., 55:1878-1883, 1987). U.S. Pat. No. 5,648,240, incorporated
herein by reference, discloses a DNA segment comprising a gene
encoding a S. aureus broad spectrum adhesin that has a molecular
weight of about 70 kDa. The adhesin is capable of binding
fibronectin or vitronectin and includes a MHC II mimicking unit of
about 30 amino acids. Further analyses of the binding specificities
of this protein reveal that it functionally resembles an MHC II
antigen in that it binds synthetic peptides. Thus, in addition to
mediating bacterial adhesion to ECM proteins, it may play a role in
staphylococcal infections by suppressing the immune system of the
host. The patent further claims a recombinant vector that includes
the specified DNA sequence, a recombinant host cell transformed
with the vector, and DNA which hybridizes with the DNA of specified
sequence. Also disclosed is a composition that includes a protein
or polypeptide encoded by the specified DNA sequence and a method
of inducing an immune response in an animal that includes
administering an immunogenic composition that includes the encoded
protein or polypeptide. A method of making a MHC II antigen protein
analog comprising the steps of inserting the specified DNA sequence
in a suitable expression vector and culturing a host cell
transformed with the vector under conditions to produce the MHC II
antigen protein analog is additionally claimed in the patent.
VII. SDR Proteins from Staphylococcus epidermidis
[0138] Staphylococcus epidermidis, a coagulase-negative bacterium,
is a common inhabitant of human skin and a frequent cause of
foreign-body infections. Pathogenesis is facilitated by the ability
of the organism to first adhere to, and subsequently to form
biofilms on, indwelling medical devices such as artificial valves,
orthopedic devices, and intravenous and peritoneal dialysis
catheters. Device-related infections may jeopardize the success of
medical treatment and significantly increase patient mortality.
Accordingly, the ability to develop vaccines that can control or
prevent outbreaks of S. epidermidis infection is of great
importance, as is the development of means that can prevent or
treat infection from a broad spectrum of bacteria, including both
coagulase-positive and coagulase negative bacteria.
[0139] Three Sdr (serine-aspartate (SD) repeat region) proteins
that are expressed by S. epidermidis have been designated as SdrF,
SdrG and SdrH, and the amino acid sequences of these proteins and
their nucleic acid sequences are disclosed in co-pending U.S.
patent application of Foster et al. which is based on U.S.
provisional application Ser. Nos. 60/098,443 and 60/117,119. All of
these applications are incorporated herein by reference.
[0140] In accordance with the present invention, the donor
selection and donor stimulation methods described herein can also
be performed with regard to the SdrF, SdrG or an SdrH protein. In
these methods, individuals may be identified and selected who have
higher than normal antibody titers to the SdrF, SdrG or an SdrH
proteins, and a donor plasma pool can be prepared which will have
higher than normal titers to one or more of these proteins.
Accordingly, donor plasma can be prepared in accordance with the
present invention which will be useful in methods to prevent or
treat infection from coagulase-negative staphylococcal infections
such as those associated with S. epidermidis.
VIII. Proteins and Peptides with Substantial Homology or Equivalent
Function to Those Described Herein
[0141] Donor plasma pools can be screened or stimulated as desired,
with full sequence proteins, peptides, protein or peptide
fragments, isolated epitopes, fusion proteins, or any alternative
which binds to the target ECM, whether in the form of a wild type,
a site-directed mutant, or a sequence which is substantially
homologous thereto.
[0142] When used in conjunction with amino acid sequences, the term
"substantially similar" means an amino acid sequence which is not
identical to published sequences, but which produces a protein or
peptide having the same functionality and activities, either
because one amino acid is replaced with another similar amino acid,
or because the change (whether it be substitution, deletion or
insertion) does not substantially effect the active site of the
protein. Two amino acid sequences are "substantially homologous"
when at least about 70%, (preferably at least about 80%, and most
preferably at least about 90 or 95%) of the amino acids match over
the defined length of the sequences.
[0143] It should also be understood that each of the MSCRAMM
polypeptides of this invention may be part of a larger protein. For
example, a ClfA polypeptide of this invention may be fused at its
N-terminus or C-terminus to a ClfB polypeptide, or to a
non-fibrinogen binding polypeptide or combinations thereof.
Polypeptides which may be useful for this purpose include
polypeptides derived any of the MSCRAMM proteins, and serotypic
variants of any of the above.
[0144] Modification and changes may be made in the structure of the
peptides of the present invention and DNA segments which encode
them and still obtain a functional molecule that encodes a protein
or peptide with desirable characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second generation
molecule. The amino acid changes may be achieved by changing the
codons of the DNA sequence, according to Table 1. In keeping with
standard polypeptide nomenclature (J. Biol. Chem., 243:3552-3559,
1969), abbreviations for amino acid residues are shown in Table I.
It should be understood by one skilled in the art that the codons
specified in Table 1 are for RNA sequences. The corresponding
codons for DNA have a T substituted for U.
[0145] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties which can stimulate the production of a substantially
similar antibody. It is thus contemplated by the inventors that
various changes may be made in the peptide sequences of the
disclosed compositions, corresponding DNA sequences which encode
said peptides or antibodies against said peptides without
appreciable loss of the biological utility or activity of the donor
plasma pool immunoglobulin that is recovered. TABLE-US-00001 TABLE
1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C
UGC UGU Aspartic acid Asp D GAC GAU GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GCG GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG GUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0146] It is understood in the art that the substitution of like
amino acids can be made effectively on the basis of hydrophilicity.
U.S. Pat. No. 4,554,101, incorporated herein by reference, states
that the greatest local average hydrophilicity of a protein, as
governed by the hydrophilicity of its adjacent amino acids,
correlates with a biological property of the protein.
[0147] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+1.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those which are within .+-.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0148] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0149] The following non-classical amino acids may be incorporated
in the peptide in order to introduce particular conformational
motifs: 1,2,3,4-tetrahydroisoquinoline-3--carboxylate (Kazmierski
et al., J. Am. Chem. Soc., 113:2275-2283, 1991);
(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,
(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine
(Kazmierski and Hruby, Tetrahedron Lett., 1991);
2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, Ph.D.
Thesis, University of Arizona, 1989);
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al,
J. Takeda Res. Labs., 43:53-76, 1989); .beta.-carboline (D and L)
(Kazmierski, Ph.D. Thesis, University of Arizona, 1988); HIC
(histidine isoquinoline carboxylic acid) (Zechel et al, Int. J.
Pep. Protein Res., 43, 1991); and HIC (histidine cyclic urea)
(Dharanipragada).
[0150] The following amino acid analogs and peptidomimetics may be
incorporated into a peptide to induce or favor specific secondary
structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a
.beta.-turn inducing dipeptide analog (Kemp et al, J. Org. Chem.,
50:5834-5838, 1985); .beta.-sheet inducing analogs (Kemp et al.,
Tetrahedron Lett., 29:5081-5082, 1988); .beta.-turn inducing
analogs (Kemp et al., Tetrahedron Lett., 29:5057-5060, 1988);
alpha-helix inducing analogs (Kemp et al., Tetrahedron Lett.,
29:4935-4938, 1988); ?-turn inducing analogs (Kemp et al., J. Org.
Chem., 54:109:115, 1989); and analogs provided by the following
references: Nagai and Sato, Tetrahedron Lett., 26:647-650 (1985);
DiMaio et al., J. Chem. Soc. Perkin Trans., p. 1687 (1989); also a
Gly-Ala turn analog (Kahn et al., Tetrahedron Lett., 30:2317,
1989); amide bond isostere (Jones et al., Tetrahedron Lett.,
29:3853-3856, 1988); tetrazol (Zabrocki et al., J. Am. Chem. Soc.,
110:5875-5880, 1988); DTC (Samanen et al., Int. J. Protein Pep.
Res., 35:501:509, 1990); and analogs taught in Olson et al., J. Am.
Chem. Sci., 112:323-333 (1990) and Garvey et al., J. Org. Chem.,
56:436 (1990). Conformationally restricted mimetics of beta turns
and beta bulges, and peptides containing them, are described in
U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.
IX. Preparation of Purified Immunoglobulin
[0151] In one embodiment, purified immunoglobulin (A, D, E, or G)
is prepared that has a high titer of antibodies to the selected
adhesins. The term "high titer" in this context means the presence
of an antibody in an amount which is 2-fold or greater, e.g., up to
10-20 more times higher than that found in a normal population of
100 random samples of blood.
[0152] The blood product can be prepared by (i) selection and
purification of the immunoglobulin of a donor which has naturally
high titers of antibodies to the selected adhesins, (ii) the
combination of donor immunoglobulin from several individuals which
have a high titer of antibodies to one or more of the selected
adhesins, to produce the desired composite profile; or (iii)
stimulation of the desired antibodies in one or more donors to form
the desired composite antibody profile by exposing the donor to the
selected antigens and obtaining blood sample of the exposed donor
after sufficient time to produce and accumulate the resulting
immunoreactive antibodies. The first two embodiments are referred
to as "donor select" programs and the third is referred to as a
"donor stimulation" program.
Donor Stimulation
[0153] Using the peptide antigens described herein, the present
invention also provides methods of stimulating high antibody levels
in a donor, which includes administering to an animal, for example
a human, a pharmaceutically-acceptable composition comprising an
immunologically effective amount of an MSCRAMM-derived peptide
composition. The composition can include partially or significantly
purified MSCRAMM-derived peptide epitopes, obtained from natural or
recombinant sources, which proteins or peptides may be obtainable
naturally or either chemically synthesized, or alternatively
produced in vitro from recombinant host cells expressing DNA
segments encoding such epitopes. Smaller peptides that include
reactive epitopes, such as those between about 30 and about 100
amino acids in length will often be preferred. The antigenic
proteins or peptides may also be combined with other agents, such
as other staphylococcal or streptococcal peptide or nucleic acid
compositions, if desired. The composition may also include
staphylococcal produced bacterial components such as those
discussed above, obtained from natural or recombinant sources,
which proteins may be obtainable naturally or either chemically
synthesized, or alternatively produced in vitro from recombinant
host cells expressing DNA segments encoding such peptides.
[0154] Further means contemplated by the inventors for generating
an immune response in an animal includes administering to the
animal, or human subject, a pharmaceutically-acceptable composition
comprising an immunologically effective amount of a nucleic acid
composition encoding a peptide epitope, or an immunologically
effective amount of an attenuated live organism that includes and
expresses such a nucleic acid composition. Antigenic functional
equivalents of the proteins and peptides described herein also fall
within the scope of the present invention. Antigenically functional
equivalents, or epitopic sequences, may be first designed or
predicted and then tested, or may simply be directly tested for
cross-reactivity.
[0155] In the case of preventing bacterial adhesion, the
preparation of epitopes which produce antibodies which inhibit the
interaction of a specific gene product or proteoglycans which are
structurally similar to the specific gene product are particularly
desirable.
[0156] The identification or design of suitable MSCRAMM epitopes,
and/or their functional equivalents, suitable for use in
immunoformulations, vaccines, or simply as antigens (e.g., for use
in detection protocols), is a relatively straightforward matter.
For example, one may employ the methods of Hopp, as enabled in U.S.
Pat. No. 4,554,101, incorporated herein by reference, that teaches
the identification and preparation of epitopes from amino acid
sequences on the basis of hydrophilicity. The amino acid sequence
of these "epitopic core sequences" may then be readily incorporated
into peptides, either through the application of peptide synthesis
or recombinant technology.
Plasmapheresis
[0157] The term plasmapheresis describes a technique in which blood
is removed from an animal, separated into its cellular and plasma
components, the cells are then returned to the animal, and the
plasma retained. Large volume plasmapheresis requires the removed
plasma to be replaced by a suitable fluid, and when this is done,
the technique is often known as plasma exchange. Any components
found in plasma can be removed by plasma exchange. Plasma exchange
is the method still in use at most blood banks and public donation
centers in the United States. Plasma extracted this way for
commercial sale is available for use in a preferred embodiment of
this invention.
[0158] During plasma donation, it is necessary to replace the fluid
taken to prevent circulatory collapse. In most circumstances, the
osmotic effect of the plasma needs to be replaced. A 5% solution of
human albumin obtained from donor blood is a safe and effective
replacement. It is standard practice in the medical community to
add 2 ml of KCl solution and 2 ml of 10% calcium gluconate solution
to the albumin. Most plasma exchange units replace every 2 liters
of plasma removed with 1.5 liters of human albumin solution and 0.5
liters of normal saline.
[0159] The methods currently in use for plasma separation are
centrifugation and filtration. The technique of U.S. Pat. No.
5,548,066 may be used to prepare the donor plasma pool if it is not
commercially available, and is incorporated by reference herein.
First, a plurality of blood donors are identified. These donors are
mature mammals, typically mammals of the same species for which the
serum will be employed. Where a specific ailment is to be treated
or prevented, such as mastitis in mammals or other diseases caused
by staphylococcal bacteria such as S. aureus, it is preferred that
the donors have been exposed either naturally or through
immunization to the causative organism or some antigenic portion
thereof. Further, to achieve a consistent serum product, it is
preferred that the donor group be relatively large. It is preferred
to use human hosts to prepare the donor plasma pools. Once the
donors have been identified, blood is drawn from the donors. Since
the serum is refined directly from the blood, it is desired to
obtain the maximum quantity of blood to thus obtain the maximum
quantity of serum. For humans, an established limit of blood is
drawn periodically over time.
[0160] It is preferred to identify and maintain a consistent donor
group by repeated drawing of smaller quantities of blood, for
example, drawing of blood once a month from humans. The frequency
of the drawing will of course influence the quantity which may be
safely drawn. In general, it is desired to draw the maximum amount
of blood over the course of time without causing detriment to the
health of the donor. This may dictate drawing small amounts with
great frequency, or the maximum amount possible at a reduced
frequency, depending upon the particular species. The blood volume
of the donor may be estimated by standard formulas available from
the Center for Disease Control.
[0161] The health of the donor is of course a consideration in this
process if long-term bleeding is desired. Before donating, the
donor will be checked for general good health, and if the donor is
in poor health the bleeding may be deferred until the next
scheduled date. Beyond this, it is preferred that long-term health
records be kept, preferably including more detailed information. In
this regard, it is noted that production quantities of the present
serum is a good indicator of the health of the donor.
[0162] Typically, the serum is separated from the blood of the
donor and consists of material from the immune system. In one
method, detailed records are kept for the amount of serum produced
from the blood as a yield percentage, such as 7 liters of serum
from 14 liters of blood provides a yield of 50%. In the preferred
method, records of the yield percentage are kept for each donor for
each bleeding. These percentages may then be used to determine if
the donor should be bled at the next scheduled time. In particular,
if the action to be taken is expressed as a function of yield
percentages, a guideline may be expressed as follows: yield
percentage </=30%, rest; 31-35%, caution; 36-59%, normal;
60-64%, caution; >/=65%, rest. As may be seen, the donor is not
bled if the serum yield is above or below the normal range. Such a
yield percentage may indicate an underlying ailment. The subject
may be bled, possibly in a reduced amount, in the caution ranges,
depending upon the donor's history and/or further examination. In
this regard, it has been found that a small percentage of
individuals consistently produce yield percentages around
60-62%.
[0163] The method of blood and plasma collection is generally
standard and well to known to those of skill in the art. Any method
can be used that achieves the desired results. Once the blood has
been collected, it is subjected to procedures for extracting the
desired components. A first important step in this process is to
permit each vessel of collected blood to sit at room temperature at
least until substantial clotting has occurred, usually one hour.
During this period the blood moves from body temperature to room
temperature, and is exposed to air. This exposure to air permits
the fibrinogen to change into fibrin, causing clotting of the
blood.
[0164] This clotting period is an important aspect of serum
retrieval. The clotting provides a rough separation of the cellular
material from the liquid. Additionally, while the exact mechanism
is not known, it is believed that the clotting period causes white
blood cells to die and, for a percentage of such cells, to burst or
rupture such that the chemical material, including antibody,
therein is released from the cells. It is believed that this
material remains within the serum and acts to provide "information"
to the immune system of the recipient of the serum. This
"information" may help to "program" white blood cells for
particular microorganisms, similar to providing them with a memory
of the microorganism, such that the white blood cells of the
recipient respond quickly, and in a manner similar to a subject
which has been vaccinated or is immune.
[0165] This period of non-refrigeration also causes a rough
filtering of the collected blood. In particular, the clotted blood
with the relatively heavy red blood cells will fall toward the
bottom of the vessel, while the liquid plasma, immunoglobulins and
chemical material will be pushed toward the top. To assist in this
process, and a process described below, it is preferred that the
collection vessel be tall and thin, having proportions similar to a
standard test tube.
[0166] The liquid portion obtained at this stage is raw serum
which, after being filtered and sterilized, can impart immunity.
Further steps are optionally carried out. However, to increase the
yield, various other steps prior to filtration are preferred.
[0167] A first of these steps, after the collected blood has had
sufficient time to clot, is refrigeration to approximately
20-60.degree. C. This refrigeration reduces the temperature of the
blood from room temperature to the refrigeration temperature. Such
cooling of course prevents growth of bacteria, mold, etc.
Additionally, during this cooling the clotted blood settles
further, and the clotted blood
[0168] contracts. This contraction (and possibly the cooling) may
cause a further percentage of the white blood cells to rupture.
Additionally, the contraction of the clotted blood serves to
express from the clot immunoglobulins and chemical materials which
have been trapped therein. This refrigeration should last at least
until the blood has achieved the refrigeration temperature, and
preferably for about 14-18 hours, or overnight.
[0169] A second preferred step is physical pressing of the clotted
blood. This pressing is believed to cause yet more rupturing of
white cells, thus yielding even more of the transfer factor.
Additionally, in a manner similar to the cooling contraction, the
pressing serves to force immunoglobulins and transfer factor from
the clot.
[0170] The preferred method of pressing is to insert a sterile
weight into the refrigerated vessel of collected blood. For
example, a cylinder having a close sliding fit within the vessel
and a weight of approximately two pounds. As may be envisioned, the
liquid material will flow about the cylinder until the cylinder has
come to rest upon the clotted blood settled at the bottom of the
vessel. It is preferred that the pressing weight be maintained in
place for about 6-24 hours.
[0171] It is noted that the pressing can serve as a first active
filtration step. The close fit of the weight serves to separate the
liquid raw serum above and the solid material below, although a
precision fit of the weight in the vessel is not required. Since
this may serve as a first, rough, filtration step, it may
conveniently be used to determine the quantity of raw serum
produced for calculation of the yield percentage. Specifically,
noting the height of the column of raw serum and knowing the
diameter of the vessel provides the volume of raw serum
produced.
[0172] At this point the filtering process proper begins. This
further processing includes filtration to remove all cellular
material. This filtration is achieved in multiple steps. The first
filtration step is a gross filtering. This may be achieved simply
by pouring the contents of the vessel into a collection vat while
holding a screen over the opening in the collection vessel. Where
the high-yield steps of refrigeration and pressing have been used,
the pressing cylinder still within the vessel may act in
conjunction with the screen to filter, and the screen may mainly
filter out the cylinder itself. Where these high-yield steps have
not been taken, a finer filter screen may be desired. The clotted
cells remaining within the vessel are properly disposed of, and the
vessel sterilized for later use.
[0173] This is a preferred point for combining the serum from
different donors. It is noted, however, that samples from multiple
donors can be combined at any point subsequent to the initial gross
filtration step.
[0174] The raw serum may still contain a large amount of cells and
cellular debris. As the next filtration step, the reclaimed liquid
is then placed into a continuous flow centrifuge. For example, the
liquid may be placed in a Sharples AS16NF continuous flow
centrifuge, which will operate at approximately 13,000 to 15,000
rpm. The liquid is drawn off during this process while yet more of
the cells and cellular debris is removed.
[0175] Following the isolation of the plasma, the antibodies are
purified away from other cell products. This can be accomplished by
a variety of protein isolation procedures, known to those skilled
in the art of immunoglobulin purification, such as ion exchange,
affinity purification, etc. Means for preparing and characterizing
antibodies are well known in the art. For example, serum samples
can be passed over protein A or protein G sepharose columns to bind
IgG (depending on the isotype). The bound antibodies are then
eluted with, e.g. a pH 5.0 citrate buffer. The elute fractions
containing the Abs, are dialyzed against an isotonic buffer.
Alternatively, the eluate is also passed over an
anti-immunoglobulin-sepharose column. The Ab is then eluted with
3.5 M magnesium chloride. Abs purified in this way can then tested
for binding activity by, for example, an isotype-specific ELISA and
immunofluorescence staining assay of the target cells.
[0176] In an alternative embodiment, the liquid is instead
subjected to a further filtration step. This further step actually
consists of several sub-steps, with the liquid being passed through
several filters of progressively finer gauge. In particular, the
liquid is passed through at least a 0.65 micron filter, then a 0.2
micron nominal filter, and then through a 0.2 micron absolute
filter. By passing the liquid through the 0.2 nominal filter first,
most of the bacteria, mold, and fibrin will be removed prior to
passing through the 0.2 absolute filter.
[0177] At this point the liquid has had essentially all solid
cellular material removed. The chemical materials and
immunoglobulins, however, remain in the liquid, which is referred
to as clarified serum.
[0178] The clarified serum can be used (after sterilization
described below) as the final serum. However, it is preferred that
the clarified serum be concentrated. This concentration reduces the
volume and thus reduces the amount of material which must be
shipped. Additionally, certain recipients, such as infant mammals,
can not accept a large quantity of medication intravenously due to
a lack of capacity. As such, concentration permits a full dosage of
the serum to be administered. The concentration is preferably
performed by repeated ultra-filtration to remove water molecules,
as is known in the art. Such filtration has a cut-off filter of
between 10,000 and 100,000 mol. wt. During this process, samples of
the clarified serum may be taken to determine if the serum has been
sufficiently concentrated. It is preferred that the final serum be
concentrated to about 2 to 6 times the clarified serum, and most
preferably 2 to 4 times.
[0179] Determination of the concentration level is made by testing
the amount of IgG (or other immunoglobulin) within the serum. An
initial test may be made of the clarified serum, and this result
compared with the tests made upon the serum during the
ultra-filtration process. For example, if the initial test results
in the clarified serum having an IgG concentration of 1 g/100 ml,
then the concentration process may be stopped when later tests
report an IgG concentration of between about 2-6 g/100 ml, and
preferably about 3 g/100 ml. The determination of the IgG amount
may be made by the radial immunodiffusion test. However, it is
preferred that serum protein electrophoresis be performed on the
whole serum to obtain an entire gamma globulin result. This is
believed to be more accurate, and provides a clear indication of
the IgG level. Once the concentration process has been completed
the concentrated unsterilized serum is bottled or packaged using
standard procedures.
[0180] Upon completion of the concentration and packaging process,
the result is unsterilized serum. The next step is to sterilize the
serum. While this sterilization is effected, it is important that
the unsterilized serum not be denatured. To provide sterilization
without denaturing, the unsterilized serum is frozen to a hard
freeze condition. For the unsterilized serum, this is approximately
-29.degree. C. (-21.degree. F.). While still frozen, the material
is then subjected to sufficient gamma irradiation that the material
is sterilized, but is not denatured. This level may vary among
various species, but may be determined without undue
experimentation. It is important that the material be sufficiently
cold (hard frozen) such that the material remains frozen during the
irradiation step, otherwise denaturing will occur. It is for this
reason that the material is frozen to the relatively low
temperature. If it is found that if the irradiation process is
sufficiently short, or refrigeration is provided during
irradiation, then a higher temperature (though still below
freezing) could be tolerated.
[0181] At this point the final serum has been obtained, although it
is frozen. The packages of the serum are thus placed in
refrigeration and allowed to thaw to the refrigeration temperature,
where they are stored until use.
[0182] After administration, the serum has been found to provide
cellular immunity similar to a vaccine, and can be used with or
without the introduction of the virulent. In general, the present
serum should provide protection against bacteria for which the
donor group has immunity. In humans, a wide variety of vaccination
uses are possible, including general vaccination for individuals
with impaired immunity, such as is caused by diabetes, and
vaccination for individuals preparing to undergo surgery due to the
of nosocomial infection. In addition to humans, the inventive serum
should also be of utility for many mammals, such as farm and
domestic mammals and humans. For cattle, one particular use would
be to avoid bovine mastitis, a common ailment which costs the dairy
industry millions of dollars per year.
X. Uses for MSCRAMM and Antibody Compositions
[0183] The immunotherapeutic product of the present invention is a
purified and concentrated extract of plasma, or serum from a
purified donor pool. The serum contains antibodies released from
the white blood cells in the extracted blood, and possibly other
chemical materials present in the extracted blood. This serum is
believed to provide information which is "read" by the immune
system of the recipient to provide an extended period of immunity,
typically on the order of six to eight weeks. Purified donor plasma
pools can be used for the treatment of wounds, for blocking protein
receptors or for immunization (vaccination).
[0184] The plasma pools comprise antibodies which are useful for
interfering with the initial physical interaction between a
pathogen and mammalian host responsible for infection, such as the
adhesion of bacteria, particularly gram positive bacteria, to
mammalian extracellular matrix proteins on in-dwelling devices or
to extracellular matrix proteins in wounds; to block
protein-mediated mammalian cell invasion; to block bacterial
adhesion between mammalian extracellular matrix proteins and
bacterial proteins that mediate tissue damage; and, to block the
normal progression of pathogenesis in infections initiated other
than by the implantation of in-dwelling devices or surgical
techniques.
[0185] In general, both poly- and monoclonal antibodies against
MSCRAMM peptides may be used in a variety of embodiments. For
example, they may be employed in antibody cloning protocols to
obtain cDNAs or genes encoding the peptides discussed herein or
related proteins. They may also be used in inhibition studies to
analyze the effects of MSCRAMM-derived peptides in cells or
animals. Anti-MSCRAMM epitope antibodies will also be useful in
immunolocalization studies to analyze the distribution of MSCRAMMs
during various cellular events, for example, to determine the
cellular or tissue-specific distribution of the MSCRAMM peptides
under different physiological conditions. A particularly useful
application of such antibodies is in purifying native or
recombinant MSCRAMMS, for example, using an antibody affinity
column. The operation of all such immunological techniques will be
known to those of skill in the art in light of the present
disclosure.
[0186] Immunological compositions, including vaccine, and other
pharmaceutical compositions containing the selected donor pool
plasma concentrate are included within the scope of the present
invention. The combination of immunoglobulins against binding
proteins, or active or antigenic fragments thereof, or fusion
proteins thereof, can be formulated and packaged, alone or in
combination with other antibodies, using methods and materials
known to those skilled in the art for vaccines. The immunological
response may be used therapeutically or prophylactically and may
provide passive immunity.
XI. Preparation of Proteins and Antibodies
[0187] The skilled reader can employ conventional molecular
biology, microbiology, and recombinant DNA techniques to prepare
the proteins, peptides, and antibody compositions described herein.
Such techniques are explained fully in the literature. See, e.g.,
Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989);
"Current Protocols in Molecular Biology" Volumes I-III (Ausubel, R.
@-I ed., 1994); "Cell Biology: A Laboratory Handbook" Volumes I-III
(J. E. Celis, ed., 1994); "Current Protocols in Immunology" Volumes
I-III ([Coligan, J. E., ed., 1994); "Oligonucleotide Synthesis" (M.
J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames &
S. J. Higgins eds., 1985); "Transcription And Translation" (B. D.
Hames & S. J. Higgins, eds., 1984); "Animal Cell Culture" (R.
I. Freshney, ed, (1986); "Immobilized Cells And Enzymes (IRL Press,
1986); B. Perbal, "A Practical Guide To Molecular Cloning"
(1984).
[0188] The antibody obtained through this invention may be labeled
directly with a detectable label for identification and
quantification of staphylococcal bacterial such as S. aureus, S.
epidermidis, etc. Labels for use in immunoassays are generally
known to those skilled in the art and include enzymes,
radioisotopes, and fluorescent, luminescent and chromogenic
substances including colored particles such as colloidal gold and
latex beads. Suitable immunoassays include enzyme-linked
immunosorbent assays (ELISA).
[0189] Alternatively, the antibody can be labeled indirectly by
reaction with labeled substances that have an affinity for
immunoglobulin, such as protein A or G or second antibodies. The
antibody may be conjugated with a second substance and detected
with a labeled third substance having an affinity for the second
substance conjugated to the antibody. For example, the antibody may
be conjugated to biotin and the antibody-biotin conjugate detected
using labeled avidin or streptavidin. Similarly, the antibody may
be conjugated to a hapten and the antibody-hapten conjugate
detected using labeled anti-hapten antibody. These and other
methods of labeling antibodies and assay conjugates are well known
to those skilled in the art. Antibodies to the binding proteins may
also be used in production facilities or laboratories to isolate
additional quantities of the protein, such as by affinity
chromatography.
[0190] In another identification embodiment, microliter plates
pre-treated with poly-L-lysine are used to bind one of the target
cells to each well, the cells are then fixed, e.g. using 1%
glutaraldehyde, and the antibodies are tested for their ability to
bind to the intact cell. In addition, FACS, immunofluorescence
staining, idiotype specific antibodies, antigen binding competition
assays, and other methods common in the art of antibody
characterization may be used in conjunction with the present
invention to identify preferred donors.
[0191] Humanized antibodies are antibodies of animal origin that
have been modified using genetic engineering techniques to replace
constant region and/or variable region framework sequences with
human sequences, while retaining the original antigen
specificity.
[0192] Such antibodies are commonly derived from rodent antibodies
with specificity against human antigens. Such antibodies are
generally useful for in vivo therapeutic applications. This
strategy reduces the host response to the foreign antibody and
allows selection of the human effector functions.
[0193] The techniques for producing humanized immunoglobulins are
well known to those of skill in the art. For example, U.S. Pat. No.
5,693,762 discloses methods for producing, and compositions of,
humanized immunoglobulins having one or more complementarily
determining regions (CDR's). When combined into an intact antibody,
the humanized immunoglobulins are substantially non-immunogenic in
humans and retain substantially the same affinity as the donor
immunoglobulin to the antigen, such as a protein or other compound
containing an epitope. Other U.S. patents, each incorporated herein
by reference, that teach the production of antibodies useful in the
present invention include U.S. Pat. No. 5,565,332, which describes
the production of chimeric antibodies using a combinatorial
approach; U.S. Pat. No. 4,816,567 which describes recombinant
immunoglobin preparations and U.S. Pat. No. 4,867,973 which
describes antibody-therapeutic agent conjugates.
[0194] U.S. Pat. No. 5,565,332 describes methods for the production
of antibodies, or antibody fragments, which have the same binding
specificity as a parent antibody but which have increased human
characteristics. Humanized antibodies may be obtained by chain
shuffling, perhaps using phage display technology, in as much as
such methods will be useful in the present invention the entire
text of U.S. Pat. No. 5,565,332 is incorporated herein by
reference.
XII. Production of High Titer MSCRAMM-Specific IgG from Biological
Fluids Via Affinity Purification
[0195] In accordance with the present invention, it is also
possible to utilize modes of affinity isolation and purification in
order to produce high titer MSCRAMM-specific immunoglobulins from
biological fluids such as blood or plasma. In the preferred modes
of this aspect of the invention, recombinant or wild-type/native
MSCRAMMs can be covalently coupled to a substrate or resin, such as
Sepharose.TM. or agarose, to form an affinity matrix. The MSCRAMM
affinity matrix can be used to selectively isolate antibodies from
serum, plasma, or other biological fluids. In the preferred
embodiment, the biological fluid is passed over the MSCRAMM
affinity matrix, and the matrix is then washed to remove
non-specifically bound antibodies. The washed matrix is then
subjected to conditions, such as low pH or high salt, so that
MSCRAMM specific antibodies remaining on the matrix are eluted. The
anti-MSCRAMM titer of the eluted material will be considerably
higher than that of the original biological fluid, and the eluted
material can then be utilized in the same manner as the other
donor-selected or donor-stimulated compositions of the present
invention.
XIII. Pharmaceutical Compositions
[0196] Pharmaceutical compositions for immunization of donors
containing the MSCRAMM proteins, nucleic acid molecules,
antibodies, or fragments thereof may be formulated in combination
with a pharmaceutical carrier such as saline, dextrose, water,
glycerol, ethanol, other therapeutic compounds, and combinations
thereof. The formulation should be appropriate for the mode of
administration. Suitable methods of administration include, but are
not limited to, oral, anal, vaginal, intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal and intradermal
administration.
The preferred route is by intravenous administration.
[0197] The pharmaceutical composition for treatment of any of the
conditions described herein, should comprise, in a pharmaceutically
acceptable excipient, an effective amount of immunoglobulin to
treat or prevent the target disorder.
[0198] Compositions which contain immunoglobulins as active
ingredients are well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions, however, solid forms suitable for
solution in, or suspension in, liquid prior to injection can also
be prepared. The preparation can also be emulsified. The active
therapeutic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. The therapeutic donor immunoglobulin pool compositions
are conventionally administered intravenously, as by injection of a
unit dose, for example. The term "unit dose" when used in reference
to a therapeutic composition of the present invention refers to
physically discrete units suitable as unitary dosage for humans,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect in association
with the required diluent; i.e., carrier, or vehicle.
[0199] The compositions are administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. The quantity to be administered depends on the subject to
be treated, capacity of the subject's immune system to utilize the
active ingredient, and degree of inhibition or neutralization of
MSCRAMM binding capacity desired. Precise amounts of active
ingredient required to be administered depend on the judgment of
the practitioner and are peculiar to each individual. However,
suitable dosages may range from about 0.1 to 20, preferably about
0.5 to about 10, and more preferably one to several, milligrams of
active ingredient per kilogram body weight of individual per day
and depend on the route of administration. Suitable regimes for
initial administration and booster shots are also variable, but are
typified by an initial administration followed by repeated doses at
one or more intervals by a subsequent injection or other
administration. Alternatively, continuous intravenous infusion
sufficient to maintain concentrations of ten nanomolar to ten
micromolar in the blood are contemplated.
[0200] The immunological compositions, such as vaccines, and other
pharmaceutical compositions can be used alone or in combination
with other blocking agents to protect against human and animal
infections caused by staphylococcal bacterial including S. aureus
and others. In particular, the compositions can be used to protect
humans against endocarditis or to protect humans or ruminants
against mastitis caused by staphylococcal infections. The vaccine
can also be used to protect canine and equine animals against
similar staphylococcal infections.
[0201] To enhance immunogenicity, the donor plasma pool concentrate
proteins may be conjugated to a carrier molecule. Suitable
immunogenic carriers include proteins, polypeptides or peptides
such as albumin, hemocyanin, thyroglobulin and derivatives thereof,
particularly bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and
solid phases. Other protein derived or non-protein derived
substances are known to those skilled in the art. An immunogenic
carrier typically has a molecular weight of at least 1,000 daltons,
preferably greater than 10,000 daltons. Carrier molecules often
contain a reactive group to facilitate covalent conjugation to the
hapten. The carboxylic acid group or amine group of amino acids or
the sugar groups of glycoproteins are often used in this manner.
Carriers lacking such groups can often be reacted with an
appropriate chemical to produce them. Preferably, an immune
response is produced when the immunogen is injected into animals
such as mice, rabbits, rats, goats, sheep, guinea pigs, chickens,
and other animals, most preferably mice and rabbits. Alternatively,
a multiple antigenic peptide comprising multiple copies of the
protein or polypeptide, or an antigenically or immunologically
equivalent polypeptide may be sufficiently antigenic to improve
immunogenicity without the use of a carrier.
[0202] In a preferred embodiment, a donor stimulating vaccine is
packaged for immunization by parenteral (i.e., intramuscular,
intradermal or subcutaneous) administration or nasopharyngeal
(i.e., intranasal) administration. The vaccine is most preferably
injected intramuscularly into the deltoid muscle. The vaccine is
preferably combined with a pharmaceutically acceptable carrier to
facilitate administration. The preferred carrier is usually water
or a buffered saline, with or without a preservative. The vaccine
may be lyophilized for resuspension at the time of administration
or in solution.
[0203] The carrier to which the protein may be conjugated may also
be a polymeric delayed release system. Synthetic polymers are
particularly useful in the formulation of a vaccine to effect the
controlled release of antigens. For example, the polymerization of
methyl methacrylate into spheres having diameters less than one
micron has been reported by Kreuter, J., "Microcapsules and
Nanoparticles in Medicine and Pharmacology," M. Donbrow, Ed., CRC
Press, p. 125-148.
[0204] The amount of immunogen composition used in the production
of the polyclonal antibodies varies upon the nature of the
immunogen, as well as the animal used for immunization. The
preferred dose for human administration is from 0.01 mg/kg to 10
mg/kg, preferably approximately 1 mg/kg. Based on this range,
equivalent dosages for heavier body weights can be determined. The
dose should be adjusted to suit the individual to whom the
composition is administered and will vary with age, weight and
metabolism of the individual. The vaccine may additionally contain
stabilizers such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury
sodium salt) (Sigma Chemical Company, St. Louis, Mo.) or
physiologically acceptable preservatives.
[0205] The production of polyclonal antibodies may be monitored by
sampling blood of the immunized animal at various points following
immunization. A second, booster injection, also may be given. The
process of boosting and titering is repeated until a suitable titer
is achieved. When a desired level of immunogenicity is obtained,
the process may continue.
[0206] The compositions preferably further comprise an adjuvant.
Many adjuvants are known for use in vaccinations in animals and are
readily adapted to this composition. At this time, the only
adjuvant widely used in humans has been alum (aluminum phosphate or
aluminum hydroxide). Saponin and its purified component Quil A,
Freund's complete adjuvant and other adjuvants used in research and
veterinary applications have toxicities which limit their potential
use in human vaccines.
[0207] The isolated peptide can be linked to a selected amino acid
sequence to make a fusion protein. As a nonlimiting example, a
fusion protein can be made that comprises at least a first peptide
of a fibronectin binding domain of fibronectin binding protein
operatively linked to a selected amino acid sequence, wherein the
first peptide does not specifically bind to fibronectin. In
preferred aspects, the first peptide is linked to a selected
carrier molecule or amino acid sequence, including, but not limited
to, keyhole limpet hemocyanin (KLH) and bovine serum albumin
(BSA).
[0208] One of the important features provided by the donor
stimulation embodiment of the present invention is a polyclonal
sera that is relatively homogenous with respect to the specificity
of the antibodies therein. Typically, polygonal antisera is derived
from a variety of different "clones," i.e., B-cells of different
lineage. Monoclonal antibodies, by contrast, are defined as coming
from antibody-producing cells with a common B-cell ancestor, hence
their "mono" clonality.
[0209] When peptides are used as antigens to stimulate the
production of polyclonal sera, one expects considerably less
variation in the clonal nature of the sera than if a whole antigen
were employed. Unfortunately, if incomplete fragments of an epitope
are presented, the peptide may very well assume multiple (and
probably non-native) conformations. As a result, even short
peptides can produce polyclonal antisera with relatively plural
specificities and, unfortunately, an antisera that does not react
or reacts poorly with the native molecule.
[0210] Polyclonal antisera according to the present invention is
produced against peptides that are predicted to comprise whole,
intact epitopes. It is believed that these epitopes are, therefore,
more stable in an immunologic sense and thus express a more
consistent immunologic target for the immune system. Under this
model, the number of potential B-cell clones that will respond to
this peptide is considerably smaller and, hence, the homogeneity of
the resulting sera will be higher. In various embodiments, the
present invention provides for polyclonal antisera where the
clonality, i.e., the percentage of clone reacting with the same
molecular determinant, is at least 80%. Even higher clonality--90%,
95% or greater--is contemplated.
XIV. Kits
[0211] This invention also includes a kit for the identification of
blood or plasma with high titers of desired antibodies. The
preferred kit contains sufficient antigen to bind substantially all
of the antibody in the sample in about ten minutes or less, or
sufficient antibody which can target an antibody in the sample that
is to be detected. The antigen or antibody in the kit, e.g., any of
the MSCRAMMs or their binding domains as described above, is
preferably immobilized on a solid support, and can be labeled with
a detectable agent such as those described above or commonly known
in the art. The kit optionally contains a means for detecting the
detectable agent. If the antigen or antibody in the kit is labeled
with a fluorochrome or radioactive label, no means for detecting
the agent will typically be provided, as the user will be expected
to have the appropriate spectrophotometer, scintillation counter,
or microscope. If the detectable agent is an enzyme, a means for
detecting the detectable agent can be supplied with the kit, and
would typically include a substrate for the enzyme in sufficient
quantity to detect all of the antigen-antibody complex. One
preferred means for detecting a detectable agent is a substrate
that is converted by an enzyme into a colored product. A common
example is the use of the enzyme horseradish peroxidase with
2,2'-azino-di-[3-ethyl-benzothiazoline sulfonate] (ABTS).
[0212] The invention includes a method for detecting biological
samples with an elevated titer of antibodies to selected
staphylococcal MSCRAMMs. As used herein the term biological sample
refers to a sample of tissue or fluid isolated from a host,
typically a human, including, but not limited to, plasma or serum.
To confirm that a factor within donor plasma is immunologically
cross-reactive with one or more epitopes of the disclosed peptides
is a straightforward matter. This can be readily determined using
specific assays, e.g., of a single proposed epitopic sequence, or
using more general screens, e.g., of a pool of randomly generated
synthetic peptides or protein fragments. The screening assays may
be employed to identify-either equivalent antigens or
cross-reactive antibodies. In any event, the principle is the same,
i.e., based upon competition for binding sites between antibodies
and antigens.
[0213] Any test which measures the binding of an antigen to an
antibody can be used to evaluate the level of antigen or antibody
in the host's biological sample according to the present invention.
A number of other such tests are known and commonly used
commercially.
[0214] Immunocytochemistry and immunohistochemistry are techniques
that use antibodies to identify antigens on the surface of cells in
solution, or on tissue sections, respectively. Immunocytochemistry
is used to quantitate individual cell populations according to
surface markers. Immunohistochemistry is used to localize
particular cell populations or antigens. These techniques are also
used for the identification of autoantibodies, using tissues or
cells that contain the presumed autoantigen as substrate. The
antibodies are usually identified using enzyme-conjugated
antibodies to the original antibody, followed by a chromogen, which
deposits an insoluble colored end product on the cell or
tissue.
[0215] Another common method of evaluation is a radioimmunoassay,
in which radiolabeled reagents are used to detect the antigen or
antibody. Antibody can be detected using plates sensitized with
antigen. The test antibody is applied and detected by the addition
of a radiolabeled ligand specific for that antibody. The amount of
ligand bound to the plate is proportional to the amount of test
antibody. This test can be reversed to test for antigen. Variations
of radioimmunoassays are competition RIA, direct binding RIA,
capture RIA, sandwich RIA, and immunoradiometric assay (RMA).
[0216] Enzyme linked immunoabsorbent assays (ELISA) are a widely
used group of techniques for detecting antigen and antibodies. The
principles are analogous to those of radioimmunoassays except that
an enzyme is conjugated to the detection system rather than a
radioactive molecule. Typical enzymes used are peroxidase, alkaline
phosphatase and 2-galactosidase. These can be used to generate
colored reaction products from colorless substrates. Color density
is proportional to the amount of reactant under investigation.
These assays are more convenient than RIA, but less sensitive.
[0217] The Western blotting (immunoblotting) method is used to
characterize unknown proteins. Components of the biological sample
are separated by gel electrophoresis. SDS gels separate according
to molecular weight and IEF gels separate the samples according to
charge characteristics. The separated proteins are transferred to
membranes (blotted) and identified by immunocytochemistry.
[0218] Less often used but suitable methods of evaluation include
the Farr assay (in which radiolabeled ligands bind to and detect
specific antibody in solution which are precipitated and
quantified), precipitin reactions (in which antibodies and antigens
crosslink into large lattices to form insoluble immune complexes;
only works if antigen and antibody are present in sufficient
amounts, at near equivalence, and when there are enough epitopes
available to form a lattice); nephelometry (measures immune
complexes formed in solution by their ability to scatter light);
immunodiffusion (detects antigens and antibodies in agar gels);
counter-current electrophoresis (similar to immunodiffusion, except
that an electric current is used to drive the antibody and antigen
together; useful for low concentrations of antigen or antibody);
single radial immunodiffusion (SRID)(quantitates antigens by
allowing them to diffuse outward from a well into an antibody
containing gel; technique can be reversed by diffusing unknown
antibody solutions into an antigen-containing well); rocket
electrophoresis (similar to SRID, except that the test antigen is
moved into the gel by an electric field); and immunofluorescence
(similar to immunochemistry, except that it used fluorescence
rather than enzyme conjugates). The antibody used to contact the
sample of body fluid is preferably immobilized onto a solid
substrate. The antibody can be immobilized using a variety of
means, as described in Antibodies: A Laboratory Manual, cited
supra. Suitable solid substrates include those having a membrane or
coating supported by or attached to sticks, synthetic glass,
agarose beads, cups, flat packs, or other solid supports. Other
solid substrates include cell culture plates, ELISA plates, tubes,
and polymeric membranes.
[0219] Means for labeling antibodies with detectable agents are
also described in Antibodies: A Laboratory Manual. The amount of
antigen in the host biological sample can be determined by any
means associated with the selected assay. For example, the selected
immunoassay can be carried out with known increasing amounts of
antigen to produce a standard curve or color chart, and then the
amount of test antigen can be determined by comparing the result of
the test to the standard curve or chart that correlates the amount
of antigen-antibody complex with known amounts of antigen. The
amount of antigen determined to be present in the host biological
sample can be used to evaluate the patient's condition in a number
of ways. First, the level of antigen can be compared to a
population norm based on statistical data. Second, the level of
antigen can be considered in light of the patient's own history of
antigen level.
[0220] The kit can optionally contain a lysing agent that lyses
cells present in the sample of body fluid. Suitable lysing agents
include surfactants such as Tween-80, Nonidet P40, and Triton
X-100. Preferably, the lysing agent is immobilized onto the solid
support along with the antibody.
[0221] The kit can also contain a buffer solution for washing the
substrate between steps. The buffer solution is typically a
physiological solution such as a phosphate buffer, physiological
saline, citrate buffer, or Tris buffer.
[0222] The kit can optionally include different concentrations of a
preformed antigen to calibrate the assay. The kit can additionally
contain a visual or numeric representation of amounts of antigen in
a calibrated standard assay for reference purposes. For example, if
an assay is used that produces a colored product, a sheet can be
included that provides a depiction of increasing intensities
associated with differing amounts of antigen.
[0223] The kit can optionally include two antibodies in the
detection system. The first antibody which is present in small
amounts is specific for the antigen being assayed for. The second
antibody provided in higher amounts is used to detect the first
antibody. For example, a rabbit antibody can be used to detect the
LOOH/amine antigen, and then an anti-rabbit IgG antibody can be
used to detect the bound rabbit antibody. Goat antibodies and
anti-antibodies are also commonly used.
[0224] As one nonlimiting example, a kit for the detection of the
lipid peroxidation state of a patient is provided that includes a
rabbit antibody specific for desired antibody, anti-rabbit IgG
antibody in sufficient amounts to detect the bound first antibody,
an enzyme conjugated to the second antibody and a substrate for the
enzyme which changes color on exposure to the enzyme.
EXAMPLES
[0225] The following examples are included to demonstrate preferred
embodiments of the present invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples which follow represent techniques discovered by the
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
Example 1
Preparation of Prototype Four Component MSCRAMM Vaccine
[0226] A series of recombinant proteins, representing domains from
the collagen, Fn, and Fbg-binding MSCRAMMs (FIG. 1), were
overexpressed in E. coli and affinity purified by metal chelating
chromatography as previously described (see, e.g., Job et al.,
Biochemistry. 33 (20):6086-6092, 1994; Patti et al., J. Biol. Chem.
270, 12005-12011, 1995; McDevitt et al., Mol. Micro. 11
(2):237-248, 1994; Ni Eidhin et al., Infect. Immun. Submitted,
1998). Used were the following: amino acids contained in the
recombinant collagen-binding MSCRAMM expressed from CNA (M55, such
as disclosed in co-pending U.S. patent application Ser. No.
08/856,253, incorporated herein by reference); amino acids
contained in the recombinant fibrinogen-binding MSCRAMM-expressed
from clfA (Region A, such as disclosed in U.S. patent application
Ser. No. 08/293,728, incorporated herein by reference); amino acids
contained in the recombinant fibrinogen-binding MSCRAMM expressed
from clfB (Region A, such as disclosed in U.S. application Ser. No.
09/200,650, incorporated herein by reference); and amino acids
contained in the recombinant fibronectin-binding MSCRAMM (DUD4,
such as those disclosed in co-pending U.S. application Ser. No.
09/010,317, incorporated herein by reference). The recombinant
FN-binding MSCRAMM protein DUD4 was treated with formalin (5%
formalin overnight, 4.degree. C.) prior to combining it with the
M55, Region A from ClfA and Region A from ClfB.
Example 2
Example of Growing E. coli Strains for Production of Recombinant
Proteins
[0227] Overnight cultures of E. coli JM101 or TOP 3 cells
(Stratagene) harboring the recombinant plasmids were diluted 1:50
in 1 L of Luria Broth (Gibco BRL) containing 50 mg/mL ampicillin.
E. coli cells were grown until the culture reached an OD.sub.600 of
0.5-0.8. Expression of the recombinant proteins was induced by
adding IPTG to a final concentration of 0.2 mM. After a three hour
induction period, cells were collected by centrifugation,
resuspended in 15 mL of Buffer A (5 mM imidazole, 0.5 M NaCl, 20 mM
Tris-HCl, pH 7.9) and lysed by passage through a French press twice
at 20,000 lb./in.sup.2. Cell debris was removed by centrifugation
at 50,000.times.g for 10 min and the supernatant was passed through
a 0.45 .mu.M filter.
Example 3
Purification of HIS.sub.6 Containing Recombinant Proteins Expressed
from pQE-30 (Qiagen.RTM.; Qiagen Inc., Chatsworth, Calif.) or PV-4
Based Recombinant Plasmids
[0228] The recombinant proteins were purified by immobilized metal
chelate chromatography, using a column of iminodiacetic
acid/Sepharose.RTM. 6B Fast Flow (Sigma, St. Louis, Mo.) charged
with Ni.sup.2+; (Porath et al. 1975; Hochuli et al. 1988). The
HIS.sub.6 tagged proteins were purified by immobilized metal
chelate affinity chromatography. More specifically, a column
containing iminodiacetic acid Sepharose.RTM. 6B FF, connected to a
FPLC.RTM. system (Pharmacia), was charged with 150 mM Ni.sup.++ and
equilibrated with buffer A (5 mM imidazole, 0.5 M NaCl, 20 mM Tris,
pH 7.9). After equilibration, the bacterial supernatant was applied
to the column and the column was washed with 10 bed volumes of
buffer A. Subsequently, the column was eluted with buffer B (200 mM
imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). The eluate was
monitored for protein by the absorbance at 280 nm and peak
fractions were analyzed by SDS-PAGE. Endotoxin was removed from the
purified recombinant proteins by detergent extraction with 1%
Triton X-114 followed by metal chelate affinity chromatography and
passage through a polymyxcin B-sepharose column. The level of
endotoxin was quantitated using a chromogenic Limulus Amebocyte
Lysate (BioWhittaker, Walkersville, Md.) assay.
Example 4
Immunization of Animals with Four Component MSCRAMM
Vaccine--MSCRAMM IV
Rhesus Monkeys:
[0229] 100 .mu.g of M55 (1 EU/mg), ClfA (2.5 EU/mg), ClfB (<1.0
EU/mg), and DUD4 (<10 EU/mg) were mixed together to form the
MSCRAMM IV vaccine. The cocktail was mixed with TiterMax.TM. Gold
(CytRX, Norcross, Ga.) in a 1:1 ratio. Two female rhesus monkeys,
ID#495Z & 664U (.sup..about.9.4 kg), were vaccinated
intramuscularly (IM) in the hind quadricep with 200 .mu.l of the
vaccine. Twenty-eight days later the two monkeys were boosted IM
with 200 .mu.l of the same vaccine formulation. Two additional
female monkeys, ID#215W & 203U (.sup..about.8.0 kg), were
immunized with the MSCRAMM IV that was compounded in a 1:1 ratio
with aluminum hydroxide (2% Alhydrogel; Superfos, Denmark).
Twenty-eight days later the two monkeys were boosted IM with 200
.mu.l of the same vaccine formulation.
[0230] The clinical regimen followed is described below:
TABLE-US-00002 Day 0 15 ml pre-immunization plasma sample, complete
blood chemistry Day 1 Vaccinate IM hind quadricep with 0.2 ml
MSCRAMM IV (100 .mu.g), injection site exam, temperature recorded
Day 7 Liver panel, temperature recorded, injection site exam Day 14
15 ml plasma sample Day 21 15 ml plasma sample Day 28 Complete
blood chemistry, temperature recorded 15 ml plasma sample, boost
with IM injection of 0.2 ml MSCRAMM IV (100 .mu.g) Day 30 Liver
panel, temperature recorded, injection site exam Day 35 Liver
panel, temperature recorded, injection site, 15 ml plasma sample
Day 42 15 ml plasma sample Day 49 15 ml plasma sample Day 106 15 ml
plasma sample
[0231] All 4 animals seroconverted following the initial
immunization. Antibody levels >3 times above background could be
detected by ELISA 106 days after the primary vaccination. The four
animals received another booster immunization in the 21.sup.st week
of the study. Each animal was given a booster of four subcutaneous
injections of 125 .mu.l of the vaccine for a total booster of 600
.mu.l of the vaccine. Antibody levels at least 3 times above
background, and as much as 15 times above background, could be
detected by ELISA 189 days after the primary vaccination. See FIG.
2. No adverse injection site reactions were detected by direct
observation by veterinarians. In addition, liver enzyme profiles,
CBC, and hematology profiles were within the normal range for
rhesus monkeys.
Example 5
Analysis of Plasma Samples from the Vaccinated Monkeys were
Analyzed by ELISA
[0232] Immulon-2 microtiter plates (Dynex Technologies, Chantilly,
Va.) were coated overnight at 4.degree. C. with 10 .mu.g/ml (50
.mu.l) of the collagen binding MSCRAMM (M55), fibrinogen binding
MSCRAMM (clfA; pCF44), fibrinogen binding MSCRAMM (ClfB; Region A),
and the fibronectin binding MSCRAMM (DUD4). Fifty microliters of
the diluted plasma samples were added to the MSCRAMM coated wells
and incubated for 1 hr at room temperature. Wash buffer consisting
of PBS containing 0.05% vol/vol Tween-20, a blocking solution of 1%
wt/vol BSA, 0.05% Tween-20 in PBS, and antibody dilution buffer
consisting of PBS containing 0.1% BSA, 0.05% Tween-20. Incubation
with primary and secondary antibodies was for 60 min at 25.degree.
C. The secondary antibody was alkaline phosphatase-conjugated goat
anti-monkey immunoglobulin G, (Rockland, Gilbertsville, Pa.),
diluted 3500-fold in antibody dilution buffer. ELISA plates were
developed for 30 min at 37.degree. C. with 1 mg/ml p-nitrophenyl
phosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl.sub.2, pH 9.8,
and quantified at 405 nm on a Perkin Elmer HTS 7000 Bio-Assay
reader. Each plasma sample was diluted 100-fold in phosphate
buffered saline, containing 0.05% Tween 20, 0.1% BSA, pH 7.4. ELISA
data are shown in FIG. 2.
Example 6
Inhibition Assays
[0233] Methicillin resistant S. aureus strain 601 (Smeltzer, M. S.,
Gene. 196:249-159, 1997) was cultured under constant rotation for
15 h at 37.degree. C. in BHI broth. A 1:100 dilution of the
overnight culture was made into BHI and the bacteria were grown at
37.degree. C. until mid exponential phase. The bacteria were
harvested by centrifugation, washed three times in sterile PBS, pH
7.4, and then resuspended in a carbonate buffer (50 mM NaHCO.sub.3,
pH 8.5). The bacteria were mixed with 1 mg/ml FITC (Sigma; F-7250)
in 50 mM NaHCO.sub.3, pH 8.5 and incubated end-over-end in the dark
for 1 hr at 25.degree. C. The FITC labeling reaction was stopped by
centrifugation of the bacterial cells and removing the supernatant
containing the unreacted FITC. The labeled bacteria were washed
three times in PBS to remove unincorporated FITC, resuspended in
PBS, adjusted to .sup..about.1.times.10.sup.8 cfu/ml and stored at
-20.degree. C. in PBS, pH 7.4.
Example 7
Purification of IgG from Immunized Monkeys
[0234] IgG was purified from the monkey plasma by affinity
chromatography on PROSEP.RTM.-A high capacity resin (Bioprocessing
Inc., Princeton, N.J.). Briefly, the plasma was thawed and passed
through 0.45.mu. filter. The plasma was applied to a benchtop
column containing PROSEP.RTM.-A high capacity resin. The unbound
material was removed by washing the column extensively with PBS.
The IgG was eluted from the column with 0.1 M sodium citrate, pH
3.0. The pH of eluted IgG was immediately neutralized to pH 6.8-7.4
by the addition of 1M Tris, pH 9.0. The IgG was then dialyzed into
PBS, pH 7.4, concentrated and filter sterilized. The concentration
of the purified IgG was determined by absorbance at 280 nm.
Example 8
Competitive Inhibition ELISA
[0235] Costar 96 well black plates were coated overnight at
4.degree. C. or at room temperature for 2 hr with a 10 .mu.g/ml
solution of matrix components consisting of bovine collagen, human
fibrinogen, and bovine fibronectin in PBS, pH 7.4. The matrix
protein coated plates were washed three times with PBS, 0.05% Tween
20 and then blocked with PBS, 1% BSA. The blocked plates were
washed three times with PBS, 0.05% Tween 20. A 500 .mu.l aliquot of
FITC-labeled S. aureus cells were mixed with an increasing amount
of purified monkey IgG in PBS, 0.05% Tween 20, 0.1% BSA. The
labeled cells and IgG were mixed on an end-over-end shaker for 1 hr
at 25.degree. C. Fifty .mu.l of the labeled cells/IgG mixture was
added to each well on the microtiter plate and incubated at
25.degree. C. on a rocker platform. The wells were washed three
times with PBS, 0.05% Tween 20. The amount of bacteria bound to the
immobilized matrix proteins was determined on a Perkin Elmer HTS
7000 Bio-Assay reader with the excitation filter set at 485 nm and
the emission filter set at 535 nm. Data are shown in FIGS. 3-5.
Example 9
Animal Model of Sepsis
[0236] Using a mouse model of sepsis (Bremell, T. A., et al.,
Infect. Immun. 62 (7):2976-2985, 1992) we have demonstrated that
passive immunization with IgG purified from rhesus monkeys
immunized with the MSCRAMM IV can protect mice against sepsis
induced death. Naive male NMRI mice 5-8 weeks old were passively
immunized i.p. on day -1 with 20 mg of either purified IgG from
rhesus monkeys immunized with MSCRAMM IV (n=12), or IgG from
non-immunized rhesus monkeys (n=13). On day 0, the mice were
challenged i.v. with 2.4.times.10.sup.7 CFU/mouse S. aureus strain
LS-1. Mortality and weight change was monitored over the next 3
days. Three days after the inoculation 3/13 mice (13%) were dead in
the control group, compared to 0/12 mice (0%) in the control group.
Mortality in control group at day 13 was 53.8% (7/13) compared to
only 16.2% (2/12) for the MSCRAMM IV passively immunized group. The
control mice exhibited a significant decrease in their body weight
compared to MSCRAMM IV IgG passively immunized mice (28.0.+-.2.5%
vs 21.3.+-.3.1%; p<0.01).
Example 10
ELISAs
[0237] ELISAs were performed in Immulon-II 96-well microtiter
plates (Dynex Technologies, Chantilly, Va.), with wash buffer
consisting of PBS containing 0.1% vol/vol Tween-80, a blocking
solution of 1% wt/vol BSA, 0.1% Tween-80 in PBS, and antibody
dilution buffer consisting of PBS containing 0.05% Tween-80.
Incubation with primary and secondary antibodies was for 60 min at
25.degree. C. The secondary antibody was alkaline
phosphatase-conjugated goat anti-human immunoglobulin G, (Chemicon,
Temecula, Calif.), diluted 3000-fold in antibody dilution buffer.
ELISA plates were developed for 30 min at 20.degree. C. with 1
mg/ml p-nitrophenyl phosphate (Sigma) in 1 M diethanolamine, 0.5 mM
MgCl.sub.2, pH 9.8 and quantified on a Molecular Dynamics ELISA
plate reader equipped with a 405 nm filter. Each human serum sample
was diluted 100-fold in phosphate buffered saline, containing 0.05%
Tween 20, 0.1% BSA, pH 7.4. The ELISA plates were coated overnight
at 4.degree. C. with 1 .mu.g/ml (100 .mu.l) of the collagen binding
MSCRAMM (M55; Patti, J. M., et al., J. Biol. Chem. 270,
12005-12011, 1995), fibrinogen binding MSCRAMM (clfA; pCF44;
McDevitt, D., et al., Mol. Micro. 11 (2):237-248, 1994), fibrinogen
binding MSCRAMM (clfB; Region A domain; Ni Edhin, D., et al.,
Infect. Immun. Submitted, 1998) and the fibronectin binding MSCRAMM
(DUD4; Joh, H. J., et al., Biochemistry. 33 (20):6086-6092, 1994).
One hundred microliters of the diluted serum samples were added to
the MSCRAMM coated wells and incubated for 1 hr at room
temperature.
[0238] 100 human plasma donor samples were analyzed using the
above-described ELISA protocol. Eight donors were selected as
having elevated MSCRAMM antibody titers ("MSCRAMM Selected").
Plasma units that ranged from 700 ml-850 ml, from eight donors were
pooled and the IgG purified by affinity chromatography on
PROSEP.RTM.-A high capacity resin (Bioprocessing Inc., Princeton,
N.J.). Briefly, the human plasma was thawed at 4.degree. C. for 24
hours and the units pooled into one batch. The plasma pool was
poured through cheesecloth and then filtered through 0.45.mu.
filter. The plasma was applied to a column of PROSEP.RTM.-A high
capacity resin connected to a preparative scale HPLC (Waters). The
unbound material was removed by washing the column extensively with
PBS. The IgG was eluted from the column with 0.1 M sodium citrate,
pH 3.0. The pH of eluted IgG was immediately neutralized to pH
6.8-7.4 by the addition of 1 M Tris, pH 9.0. The IgG was then
dialyzed into PBS, concentrated and filter sterilized. The
concentration of the purified IgG was determined by absorbance at
280 nm.
Example 11
Animal Model of S. aureus Infection
[0239] A rabbit model of infectious endocarditis (Perlman, B. B.,
and L. S. Freedman, Yale J. Biol. Med. 42:394-410, 1971) was used
to evaluate the prophylactic potential of the "MSCRAMM Selected"
human IgG. This model has been used over the past decade to
investigate the pathogenesis of endocarditis and to test a variety
of new antibiotics and vaccines. In this model, 2.5 kg rabbits
underwent a transcarotid-transaortic valvular catheterization with
an indwelling polyethylene catheter. Eight rabbits were then
treated intraperitoneally with 18 ml of the "MSCRAMM Selected"
human IgG (28 mg/ml; 504 mg total). Eight control rabbits received
18 ml sterile PBS and ten rabbits received 500 mg of normal human
IVIG (Alpha Therapeutics Veniglobulin S; Los Angeles, Calif.)
intraperitoneally. Infective endocarditis was produced 18 hours
after IgG administration by an intraperitoneal injection of
10.sup.9 cfu S. aureus strain Reynolds. The animals were followed
for 72 hours and blood samples were obtained at 12-hour intervals.
After 72 hours, the animals were euthanized and the kidneys and
valvular vegetations aseptically removed. The tissue samples were
processed for quantitative culture. Rabbits were considered
positive for endocarditis if bacteria were recovered from the
vegetations, irrespective of the bacterial density. It should be
noted that the lowest level of bacterial detection in this model is
=2 log.sub.10 cfu/g tissue. The number of organisms recovered from
the tissue sites (kidney and valvular vegetations) were
statistically compared using a two-tailed Student's t-test. P
values lower than 0.05 for individual comparisons are considered
significant. The results are shown in table 2. TABLE-US-00003 TABLE
2 MEAN FREQUENCY VEGETATION MEAN RENAL ANIMAL FREQUENCY OF OF RENAL
DENSITIES DENSITIES GROUP ENDOCARDITIS SEEDING Log.sub.10 cfu/g
.+-. SD Log.sub.10 cfu/g .+-. SD A 7/8 8/8 6.07 .+-. 3.33 4.48 .+-.
1.32 B 1/8 0/8 2.25 .+-. 0.62* 2.00 .+-. O.OO{circumflex over ( )}
C 10/10 10/10 7.42 .+-. 3.45 6.17 .+-. 2.08 Group A = PBS Group B =
MSCRAMM selected human IgG Group C = Normal IVIG (Alpha
Therapeutics Veniglobulin S) *p = 0.05 (vs. group A) {circumflex
over ( )}p = 0.05 (vs. group A), p = 0.001 (vs. group C)
Example 12
Tests Regarding ClfA and CNA Selected Human IVIG
A. Prophylactic Administration of ClfA and CNA Donor Selected
SA-IVIG IVIG
[0240] The objective of the studies described here was to determine
if passive immunization with donor selected IVIG products prepared
from human donor plasma containing high titers of antibodies
against microbial surface components recognizing adhesive matrix
molecule (MSCRAMM) proteins expressed by Staphylococcus aureus (S.
aureus) can prevent mortality caused by an antibiotic resistant S.
aureus clinical isolate in a murine septicemia model.
[0241] SA-IVIG MS502, S. aureus Immunoglobulin Intravenous.
[0242] Human IgG was purified using 15 units of plasma collected
from 7 donors that were determined to possess elevated levels of
IgG (>5 fold increase in titer compared to normal IVIG) in an
ELISA assay specific for the A domain of the ClfA MSCRAMM protein
expressed by S. aureus. Plasma was obtained from Serologicals, Inc.
(Clarkston, Ga.) and the IgG was purified by Cangene Corp. as a
sterile-filtered solution in 10% maltose, 0.03% polysorbate 80. The
material was reported by Cangene to contain 47.55 mg/ml IgG by
radial immunodiffusion. This material was stored at 4.degree. C.,
as directed by the manufacturer. On the day of administration,
MS502 was diluted to 40 mg/ml with 1.times.D-PBS in preparation for
an IP injection of 0.5 ml.
[0243] SA-IVIG MS503, S. aureus Immunoglobulin Intravenous.
[0244] Human IgG was purified using 12 units of plasma collected
from 5 donors that were determined to possess elevated levels of
IgG (>5 fold increase in titer compared to normal IVIG) in an
ELISA assay specific for the A domain of the CNA MSCRAMM expressed
by S. aureus. Plasma was obtained from Serologicals, Inc.
(Clarkston, Ga.) and the IgG was purified by Cangene Corp. as a
sterile-filtered solution in 10% maltose, 0.03% polysorbate 80. The
material was reported by Cangene to contain 45.41 mg/ml IgG by
radial immunodiffusion. This material was stored at 4.degree. C.,
as directed by the manufacturer. On the day of administration,
MS503 was diluted to 40 mg/ml with 1.times.D-PBS in preparation for
an IP injection of 0.5 ml.
[0245] Control, Human Immune Globulin Intravenous, Polygam.RTM.
(Baxter IVIG).
[0246] A sterile freeze-dried preparation of IgG was manufactured
from large pools of human plasma by Baxter Healthcare Corp. The
material was reconstituted in sterile water for injection according
to the manufacturer's directions (Baxter Healthcare Corp.). The 5%
solution contains 50 mg/ml total protein, 45 mg/ml IgG, 8.5 mg/ml
NaCl, 3 mg/ml human albumin, 22.5 mg/ml glycine, 20 mg/ml glucose,
2 mg/ml polyethylene glycol, 1 .mu.g/ml tri(n-butyl) phosphate, 1
.mu.g/ml octoxynol 9 and 100 .mu.g/ml polysorbate 80. Prior to
injection the stock was diluted in sterile distilled water to a
final concentration of 40 mg/ml IgG in preparation for an IP
injection of 0.5 ml.
[0247] S. aureus
[0248] S. aureus strain 601 (Smeltzer, et. al., 1996) was obtained
from Dr. M. S. Smeltzer, University of Arkansas for Medical
Sciences. This S. aureus strain was isolated from an intensive care
unit. The isolate is cephalothin, ciprofloxacin, clindamycin,
erythromycin, oxacillin (methicillin), penicillin-G and
trimethoprin-sulfamethoxazole resistant. Strain 601 S. aureus cells
taken from a frozen glycerol stock, were inoculated into a 10 ml
BHI broth culture and grown over night at 37.degree. C. Cells from
the overnight culture were diluted 1:100 in BHI broth and grown at
37.degree. C. for approximately 3 hours until the absorbance at 600
nm reached 1.8-2.0 OD units. The bacteria were pelleted by
centrifugation and resuspended in 1/5 volume of freezing medium
(1.times.D-PBS, pH 7.4; 10% DMSO; 5% BSA). A small aliquot of the
stock was plated on blood agar dishes at 10.sup.-2, 10.sup.-4 and
10.sup.-6 dilutions and cultured over night to determine the CFU
concentration of the preparation. The bacterial preparation was
stored at -86.degree. C. until used. On the day of injection, the
frozen bacterial stock was thawed and pelleted by centrifugation.
The bacteria were washed once in D-PBS and resuspended to the
appropriate concentration in D-PBS for IV injection. A portion of
the bacterial suspension was plated on blood agar dishes at
10.sup.-2, 10.sup.-4 and 10.sup.-6 dilutions and cultured over
night to determine the CFU concentration of the final injected
preparation.
[0249] Animal Sex, Species, Number, Age and Source
[0250] 56 female mice (5-6 weeks of age) were purchased from
Taconic Quality Laboratory Animals and Services for Research
(Germantown, N.Y.). Animals were allowed to acclimate for at least
14 days prior to initiation of treatment. Upon arrival, the mice
were examined, group housed (5/cage) in polycarbonate shoe box
cages with absorbent bedding. All mice were placed on a 12 hour
light-dark cycle under the required husbandry standards found in
the NIH Guide for the Care and Use of Laboratory Animals.
[0251] Identification and Randomization
[0252] All animals were uniquely identified using tail tattoos
prior to dosing. Prior to initiation of treatment, the animals were
individually weighed and their health was evaluated. Mice were
randomized and assigned to treatment groups using stratified body
weights.
[0253] Experimental Design
[0254] On Day -1, animals were treated with a single 0.5 ml IP
injection of MS502, MS503 or Baxter IVIG. On Day 0,
2.2.times.10.sup.8 CFU S. aureus were administered by a single IV
injection (0.1 ml) to all animals via the tail vein.
[0255] DATA
[0256] Mice were pre-treated by intraperitoneal (IP) injection with
20 mg of either Baxter's normal IVIG product or 20 mg SA-IVIG MS502
and 20 mg SA-IVIG MS503. MS502 was an immunoglobulin G (IgG)
preparation purified from donor plasma containing elevated titers
of antibodies recognizing the A domain of clumping factor (ClfA), a
S. aureus fibrinogen binding MSCRAMM protein. Likewise MS503 was a
high titer preparation selected for recognition of the A domain of
CNA, the collagen binding S. aureus MSCRAMM. The total IgG
concentrations of the two MSCRAMM preparations and the standard
Baxter's normal product is provided in Table 3, below. As shown
below, the high-titer MS502 sample had a total ClfA content of 2.29
Units/mg as opposed to only 0.2 Units/mg in the normal sample. The
high-titer MS503 sample had a total CNA content of 1.06 Units/mg as
opposed to only 0.2 Units/mg in the normal sample. 24 hours after
IgG administration, the mice were challenged with a single
intravenous (IV) injection of a methicillin-resistant strain of S.
aureus (Strain 601). The mice were followed for 5 days at which
point all remaining mice were sacrificed. Significant differences
in the relative survival times between treatment groups were
detected. Sixty-three percent (12/19) of the mice that received
MS502 (p=0.003 vs. control; Mantel-Cox survival analysis) survived
the bacterial challenge. Sixty-eight percent (13/19) of the mice
that received MS503 (p=0.0008 vs. control; Mantel-Cox survival
analysis) survived the bacterial challenge. Only 22% (4/18) of the
mice treated with normal human IVIG survived the entire study
period. These results clearly indicate that prophylactic
administration of ClfA and CNA donor selected SA-IVIG IVIG provides
a significant level of protection against lethal infection as
compared to a commercially available normal human IVIG product.
TABLE-US-00004 TABLE 3 Total IgG Selection on Concentration ClfA
CNA Product MSCRAMM (mg/ml) (Units/mg) (Units/mg) Normal Donor
Unselected 45.00 0.2 0.2 Pool MS502 ClfA 47.55 2.29 NT MS503 CAN
45.41 NT 1.06 NT = not tested Baxter Gammagard .RTM. IgIV
represents a normal unselected IgIV
B. Therapeutic Applications of ClfA-Selected Human SA-IVIG
[0257] SA-IVIG MS502, S. aureus Immunoglobulin Intravenous.
[0258] Human IgG was purified using 15 units of plasma collected
from 7 donors that were determined to possess elevated levels of
IgG (>5 fold increase in titer compared to normal IVIG) in an
ELISA assay specific for the A domain of the ClfA MSCRAMM protein
expressed by S. aureus. Plasma was obtained from Serologicals, Inc.
(Clarkston, Ga.) and the IgG was purified by Cangene Corp. as a
sterile-filtered solution in 10% maltose, 0.03% polysorbate 80. The
material was reported by Cangene to contain 47.55 mg/ml IgG by
radial immunodiffusion. This material was stored at 4.degree. C.,
as directed by the manufacturer. On the day of administration,
MS502 was diluted to 40 mg/ml with 1.times.D-PBS in preparation for
an IP injection of 0.5 ml.
[0259] S. aureus
[0260] S. aureus strain 601 (Smeltzer, et. al., 1996) was obtained
from Dr. M. S. Smeltzer, University of Arkansas for Medical
Sciences. This S. aureus strain was isolated from an intensive care
unit. The isolate is cephalothin, ciprofloxacin, clindamycin,
erythromycin, oxacillin (methicillin), penicillin-G and
trimethoprin-sulfamethoxazole resistant. Strain 601 S. aureus cells
taken from a frozen glycerol stock, were inoculated into a 10 ml
BHI broth culture and grown over night at 37.degree. C. Cells from
the overnight culture were diluted 1:100 in BHI broth and grown at
37.degree. C. for approximately 3 hours until the absorbance at 600
nm reached 1.8-2.0 OD units. The bacteria were pelleted by
centrifugation and resuspended in 1/5 volume of freezing medium
(1.times.D-PBS, pH 7.4; 10% DMSO; 5% BSA). A small aliquot of the
stock was plated on blood agar dishes at 10.sup.-2, 10.sup.-4 and
10.sup.-6 dilutions and cultured over night to determine the CFU
concentration of the preparation. The bacterial preparation was
stored at -86.degree. C. until used. On the day of injection, the
frozen bacterial stock was thawed and pelleted by centrifugation.
The bacteria were washed once in D-PBS and resuspended to the
appropriate concentration in D-PBS for IV injection. A portion of
the bacterial suspension was plated on blood agar dishes at
10.sup.-2, 10.sup.-4 and 10.sup.-6 dilutions and cultured over
night to determine the CFU concentration of the final injected
preparation.
[0261] Animal Sex, Species, Number, Age and Source
[0262] Female mice (5-6 weeks of age) were purchased from Taconic
Quality Laboratory Animals and Services for Research (Germantown,
N.Y.). Animals were allowed to acclimate for at least 5 days prior
to initiation of treatment. Upon arrival, the mice were examined,
group housed (5/cage) in polycarbonate shoe box cages with
absorbent bedding. All mice were placed on a 12 hour light-dark
cycle under the required husbandry standards found in the NIH Guide
for the Care and Use of Laboratory Animals.
[0263] Identification and Randomization
[0264] All animals were uniquely identified using tail tattoos
prior to dosing. Prior to initiation of treatment, the animals were
individually weighed and their health was evaluated. Mice were
randomized and assigned to treatment groups using stratified body
weights.
[0265] Experimental Design
[0266] On Day -1, animals were treated with a single 0.5 ml IP
injection of MS502 IVIG. On Day 0, 5.6.times.10.sup.7 CFU S. aureus
601 was administered by a single IV injection (0.1 ml) to all
animals via the tail vein. In addition, a group of mice received
MS502 IVIG 3 hours after the IV bacterial challenge. Control mice
were left untreated.
[0267] DATA
[0268] Mice were treated by intraperitoneal (IP) injection with 20
mg of SA-IVIG MS502 either 18 or prior or 3 hours after an IV
challenge with S. aureus 601. MS502 was an immunoglobulin G (IgG)
preparation purified from donor plasma containing elevated titers
of antibodies recognizing the A domain of clumping factor (ClfA), a
S. aureus fibrinogen binding MSCRAMM protein. The mice were
followed for 5 days at which point all remaining mice were
sacrificed. Ninety-three percent of the mice that received MS502
SA-IVIG 18 hours prior to S. aureus challenge survived. Similarly,
93% of the mice that received MS502 SA-IVIG 3 hours post bacterial
challenge survived. In contrast, only 76% of the control mice
survived the bacterial challenge. These results clearly indicate
that therapeutic administration of ClfA donor selected human
SA-IVIG provides a significant and effective treatment of
staphylococcal infection as compared to a commercially available
normal human IVIG product.
Sequence CWU 1
1
4 1 18 DNA Staphylococcus aureus MISC_FEATURE (6) and (12) n = (a
or c or g or t) 1 gaytcngayt cngayagy 18 2 5 PRT Staphylococcus
aureus MISC_FEATURE (3)..(3) Xaa can be any amino acid 2 Leu Pro
Xaa Thr Gly 1 5 3 9 PRT Staphylococcus aureus 3 Thr Tyr Thr Phe Thr
Asp Tyr Val Asp 1 5 4 6 PRT Staphylococcus aureus 4 Thr Asn Ser His
Gln Asp 1 5
* * * * *