U.S. patent application number 17/033195 was filed with the patent office on 2021-01-14 for bacteriophage lysin and antibiotic combinations against gram positive bacteria.
The applicant listed for this patent is ContraFect Corporation. Invention is credited to Han LEE, Robert C. NOWINSKI, Brent SCHNEIDER, Raymond SCHUCH, Michael WITTEKIND.
Application Number | 20210008175 17/033195 |
Document ID | / |
Family ID | 1000005106865 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210008175 |
Kind Code |
A1 |
SCHUCH; Raymond ; et
al. |
January 14, 2021 |
BACTERIOPHAGE LYSIN AND ANTIBIOTIC COMBINATIONS AGAINST GRAM
POSITIVE BACTERIA
Abstract
The present invention provides compositions and methods for
prevention, amelioration and treatment of gram positive bacteria,
particularly Staphylococcal bacteria, with combinations of lysin,
particularly Streptococcal lysin, particularly the lysin PlySs2,
and one or more antibiotic, including daptomycin, vancomycin,
oxacillin, linezolid, or related antibiotic(s).
Inventors: |
SCHUCH; Raymond; (Mountain
Lakes, NJ) ; NOWINSKI; Robert C.; (New York, NY)
; WITTEKIND; Michael; (Bainbridge Island, WA) ;
LEE; Han; (Yonkers, NY) ; SCHNEIDER; Brent;
(Glenmoore, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ContraFect Corporation |
Yonkers |
NY |
US |
|
|
Family ID: |
1000005106865 |
Appl. No.: |
17/033195 |
Filed: |
September 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14399575 |
Nov 7, 2014 |
10813983 |
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PCT/US2013/040329 |
May 9, 2013 |
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17033195 |
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61737239 |
Dec 14, 2012 |
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61644944 |
May 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 38/47 20130101; A61K 38/14 20130101; A61K 38/12 20130101; A61K
38/46 20130101; C12Y 302/01017 20130101; C12N 9/503 20130101; A61K
31/5377 20130101; A61K 31/4188 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C12N 9/50 20060101 C12N009/50; A61K 31/4188 20060101
A61K031/4188; A61K 31/5377 20060101 A61K031/5377; A61K 38/12
20060101 A61K038/12; A61K 38/47 20060101 A61K038/47; A61K 45/06
20060101 A61K045/06; A61K 38/14 20060101 A61K038/14 |
Claims
1-15. (canceled)
16. A method of enhancing the effectiveness of a gram-positive
antibiotic comprising administering the antibiotic with PlySs2
lysin comprising the amino acid sequence provided in FIG. 29 (SEQ
ID NO: 1) or variants thereof having at least 80% identity, 85%
identity, 90% identity, 95% identity or 99% identity to the
polypeptide of FIG. 29 (SEQ ID NO: 1) and effective to kill
gram-positive bacteria, whereby the antibiotic is at least 10 fold
more effective in combination with Plyss2.
17. (canceled)
18. The method of claim 16 wherein the antibody is at least 50 fold
more effective in combination with PlySs2 lysin.
19. The method of claim 16 wherein the PlySs2 lysin is at least two
fold more effective in combination with antibody.
20. The method of claim 16 wherein the PlySs2 lysin is at least
four fold more effective in combination with antibody.
21-23. (canceled)
24. A composition for use in inhibiting gram positive bacteria
selected from Staphylococcus, Streptococcus, Enterococcus and
Listeria comprising PlySs2 lysin polypeptide and one or more
antibiotic.
25. The composition of claim 24 wherein the antibiotic is selected
from vancomycin or a related antibiotic, linezolid or a related
antibiotic, oxacillin or a related antibiotic and daptomycin or a
related antibiotic.
26. The composition of claim 24 wherein the antibiotic is
daptomycin, vancomycin, oxacillin or linezolid.
27. The composition of claim 24 wherein the dose of antibiotic is
at least X fold lower than the ordinary clinical dose.
28. A method of killing Staphylococcus and/or Streptococcus
bacteria comprising: contacting the bacteria with a lysin
polypeptide in combination with an antibiotic, wherein the lysin
polypeptide is effective to kill Staphylococcus and/or
Streptococcus bacteria, wherein the lysin polypeptide comprises SEQ
ID NO: 1 or a variant thereof having at least 80% identity to the
amino acid of SEQ ID NO: 1 and effective to kill the one or more of
Staphylococcus and Streptococcus bacteria, wherein an amount of the
lysin polypeptide effective to kill the Staphylococcus and/or
Streptococcus bacteria in the presence of the antibiotic is less
than in the absence of the antibiotic, and wherein an amount of
antibiotic effective to kill the Staphylococcus and/or the
Streptococcus bacteria in the presence of the lysin polypeptide is
less than in the absence of the lysin polypeptide.
29. The method of claim 28, wherein the antibiotic and the lysin
polypeptide are administered sequentially.
30. The method of claim 28, wherein the antibiotic and the lysin
polypeptide are administered concurrently.
31. The method of claim 28, wherein the lysin polypeptide is
administered in a single dose.
32. The method of claim 28, wherein the lysin polypeptide is
administered in multiple doses.
33. The method of claim 28, wherein both the antibiotic and the
lysin polypeptide are administered at doses below the minimal
inhibitory concentration (MIC) dose.
34. The method of claim 28, wherein a dose of the antibiotic is
lower than the minimal inhibitory concentration (MIC) dose.
35. The method of claim 1, wherein the antibiotic is a
glycopeptide.
36. The method of claim 35, wherein the glycopeptide is
vancomycin.
37. The method of claim 1, wherein the antibiotic is a
lipopeptide.
38. The method of claim 37, wherein the lipopeptide is
daptomycin.
39. The method of claim 1, wherein the antibiotic is a beta lactam
penicillin.
40. The method of claim 39, wherein the beta lactam penicillin is
penicillin.
41. The method of claim 1, wherein the antibiotic is an
oxazolidinone.
42. The method of claim 41, wherein the oxazolidinone is
linezolid.
43. The method of claim 28, wherein the Staphylococcus bacteria
comprise Staphylococcus aureus.
44. The method of claim 43, wherein the Staphylococcus aureus is
methicillin resistant Staphylococcus aureus (MRSA).
45. The method of claim 28, wherein the PlySs2 binding domain
variant has at least 90% identity to the amino acid sequence of SEQ
ID NO: 1.
46. The method of claim 28, wherein the PlySs2 binding domain
comprises SEQ ID NO: 1.
47. The method of claim 28, wherein the lysin polypeptide and the
antibiotic are administered to a subject having bacteremia.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to prevention,
amelioration and treatment of gram positive bacteria, including
Staphylococcal bacteria, with combinations of lysin, particularly
Streptococcal lysin, particularly the lysin PlySs2, and one or more
antibiotic.
BACKGROUND OF THE INVENTION
[0002] The development of drug resistant bacteria is a major
problem in medicine as more antibiotics are used for a wide variety
of illnesses and other conditions. The use of more antibiotics and
the number of bacteria showing resistance has prompted longer
treatment times. Furthermore, broad, non-specific antibiotics, some
of which have detrimental effects on the patient, are now being
used more frequently. A related problem with this increased use is
that many antibiotics do not penetrate mucus linings easily.
[0003] Gram-positive bacteria are surrounded by a cell wall
containing polypeptides and polysaccharide. Gram-positive bacteria
include but are not limited to the genera Actinomyces, Bacillus,
Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus,
Mycobacterium, Corynebacterium, and Clostridium. Medically relevant
species include Streptococcus pyogenes, Streptococcus pneumoniae,
Staphylococcus aureus, and Enterococcus faecalis. Bacillus species,
which are spore-forming, cause anthrax and gastroenteritis.
Spore-forming Clostridium species are responsible for botulism,
tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium
species cause diphtheria, and Listeria species cause
meningitis.
[0004] Novel antimicrobial therapy approaches include enzyme-based
antibiotics ("enzybiotics") such as bacteriophage lysins. Phages
use these lysins to digest the cell wall of their bacterial hosts,
releasing viral progeny through hypotonic lysis. A similar outcome
results when purified, recombinant lysins are added externally to
Gram-positive bacteria. The high lethal activity of lysins against
gram-positive pathogens makes them attractive candidates for
development as therapeutics (Fischetti, V. A. (2008) Curr Opinion
Microbiol 11:393-400; Nelson, D. L. et al (2001) Proc Natl Acad Sci
USA 98:4107-4112). Bacteriophage lysins were initially proposed for
eradicating the nasopharyngeal carriage of pathogenic streptococci
(Loeffler, J. M. et al (2001) Science 294: 2170-2172; Nelson, D. et
al (2001) Proc Natl Acad Sci USA 98:4107-4112). Lysins are part of
the lytic mechanism used by double stranded DNA (dsDNA) phage to
coordinate host lysis with completion of viral assembly (Wang, I.
N. et al (2000) Annu Rev Microbiol 54:799-825). Lysins are
peptidoglycan hydrolases that break bonds in the bacterial wall,
rapidly hydrolyzing covalent bonds essential for peptidoglycan
integrity, causing bacterial lysis and concomitant progeny phage
release.
[0005] Lysin family members exhibit a modular design in which a
catalytic domain is fused to a specificity or binding domain
(Lopez, R. et al (1997) Microb Drug Resist 3:199-211). Lysins can
be cloned from viral prophage sequences within bacterial genomes
and used for treatment (Beres, S. B. et al (2007) PLoS ONE
2(8):1-14). When added externally, lysins are able to access the
bonds of a Gram-positive cell wall (Fischetti, V. A. (2008) Curr
Opinion Microbiol 11:393-400). Bacteriophage lytic enzymes have
been established as useful in the assessment and specific treatment
of various types of infection in subjects through various routes of
administration. For example, U.S. Pat. No. 5,604,109 (Fischetti et
al.) relates to the rapid detection of Group A streptococci in
clinical specimens, through the enzymatic digestion by a
semi-purified Group C streptococcal phage associated lysin enzyme.
This enzyme work became the basis of additional research, leading
to methods of treating diseases. Fischetti and Loomis patents (U.S.
Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use of a
lysin enzyme produced by group C streptococcal bacteria infected
with a C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and
Loomis) discloses a composition for dermatological infections by
the use of a lytic enzyme in a carrier suitable for topical
application to dermal tissues. U.S. Pat. No. 6,254,866 (Fischetti
and Loomis) discloses a method for treatment of bacterial
infections of the digestive tract which comprises administering a
lytic enzyme specific for the infecting bacteria. The carrier for
delivering at least one lytic enzyme to the digestive tract is
selected from the group consisting of suppository enemas, syrups,
or enteric coated pills. U.S. Pat. No. 6,264,945 (Fischetti and
Loomis) discloses a method and composition for the treatment of
bacterial infections by the parenteral introduction
(intramuscularly, subcutaneously, or intravenously) of at least one
lytic enzyme produced by a bacteria infected with a bacteriophage
specific for that bacteria and an appropriate carrier for
delivering the lytic enzyme into a patient.
[0006] Phage associated lytic enzymes have been identified and
cloned from various bacteriophages, each shown to be effective in
killing specific bacterial strains. U.S. Pat. Nos. 7,402,309,
7,638,600 and published PCT Application WO2008/018854 provides
distinct phage-associated lytic enzymes useful as antibacterial
agents for treatment or reduction of Bacillus anthracia infections.
U.S. Pat. No. 7,569,223 describes lytic enzymes for Streptococcus
pneumoniae. Lysin useful for Enterococcus (E. faecalis and E.
faecium, including vancomycin resistant strains) are described in
U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutant Ply GBS
lysins highly effective in killing Group B streptococci. A chimeric
lysin denoted ClyS, with activity against Staphylococci bacteria,
including Staphylococcus aureus, is detailed in WO 2010/002959.
[0007] Based on their rapid, potent, and specific cell
wall-degradation and bactericidal properties, lysins have been
suggested as antimicrobial therapeutics to combat Gram-positive
pathogens by attacking the exposed peptidoglycan cell walls from
outside the cell (Fenton, M et al (2010) Bioengineered Bugs 1:9-16;
Nelson, D et al (2001) Proc Natl Acad Sci USA 98:4107-4112).
Efficacies of various lysins as a single agents have been
demonstrated in rodent models of pharyngitis (Nelson, D et al
(2001) Proc Natl Acad Sci USA 98:4107-4112), pneumonia (Witzenrath,
M et al (2009) Crit Care Med 37:642-649), otitis media (McCullers,
J. A. et al (2007) PLOS pathogens 3:0001-0003), abscesses
(Pastagia, M et al Antimicrobial agents and chemotherapy
55:738-744) bacteremia (Loeffler, J. M. et al (2003) Infection and
Immunity 71:6199-6204), endocarditis (Entenza, J. M. et al (2005)
Antimicrobial agents and chemotherapy 49:4789-4792), and meningitis
(Grandgirard, D et al (2008) J Infect Dis 197:1519-1522). In
addition, lysins are generally specific for their bacterial host
species and do not lyse non-target organisms, including human
commensal bacteria which may be beneficial to gastrointestinal
homeostasis (Blaser, M. (2011) Nature 476:393-394; Willing, B. P.
et al (2011) Nature reviews. Microbiology 9:233-243)
[0008] Antibiotics in clinical practice include several which
commonly affect cell wall peptidoglycan biosynthesis in gram
positive bacteria. These include glycopeptides, which as a class
inhibit peptidoglycan synthesis by preventing the incorporation of
N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide
subunits into the peptidoglycan matrix. Available glycopeptides
include vancomycin and teicoplanin, with vancomycin a primary drug
of choice and clinical application in bacteremia, particularly
Staphylococcal infections. Penicillins act by inhibiting the
formation of peptidoglycan cross-links. Common penicillins include
oxacillin, ampicillin and cloxacillin. Linezolid (Zyvox) is a
protein synthesis inhibitor and in a class of antibacterials called
oxazolidinones (Ford C W et al (1996) Antimicrob Agents Chemoth
40(6):1508-1513; Swaney S M et al (1998) Antimicrob Agents Chemoth
42(12):3251-3255; U.S. Pat. No. 6,444,813).
[0009] Daptomycin (Cubicin), also denoted LY 146032, is a
lipopeptide antibacterial agent consisting of a 13-member amino
acid peptide linked to a 10-carbon lipophilic tail (Miao V et al
(2005) Microbiology 151(Pt5):1507-1523; Steenbergen J N et al
(2005) J Antimicrob Chemother 55(3):283-288; and described in U.S.
Pat. No. 5,912,226). This structure results in a novel mechanism of
action, the disruption of the bacterial membrane through the
formation of transmembrane channels, which cause leakage of
intracellular ions leading to depolarizing the cellular membrane
and inhibition of macromolecular synthesis. Daptomycin's spectrum
of activity is limited to Gram-positive organisms, including a
number of highly resistant species (methicillin-resistant S. aureus
(MRSA), vancomycin intermediate-sensitive S. aureus (VISA),
vancomycin-resistant S. aureus (VRSA), vancomycin-resistant
Enterococcus (VRE)). In studies it appears to be more rapidly
bactericidal than vancomycin. Its approved dosing regimen is 4
mg/kg IV once daily. Dose adjustment is necessary in renal
dysfunction. Daptomycin's primary toxicity is reversible
dose-related myalgias and weakness. Daptomycin has been approved
for the treatment of complicated skin and soft tissue infections
caused by gram positive bacteria, Staphylococcus aureus bacteremia
and right-sided S. aureus endocarditis. Trials assessing
daptomycin's efficacy in treating complicated urinary tract
infections and endocarditis/bacteremia are ongoing. Its approved
dosing regimen is 4 mg/kg IV once daily. Dose adjustment is
necessary in renal dysfunction. Daptomycin's primary toxicity is
reversible dose-related myalgias and weakness. Resistance to
daptomycin has been encountered both in vitro and in vivo after
exposure to daptomycin. The mechanism(s) of resistance are not
fully defined but likely relate to alterations of the cellular
membrane. Multiple passages of Staphylococci and Enterococci in
subinhibitory drug concentrations resulted in MIC increases in a
stepwise fashion. Daptomycin binds avidly to pulmonary surfactant
and cannot be effectively used in treatment of pneumonia (Baltz R H
(2009) Curr Opin Chem Biol 13(2):144-151).
[0010] The broad spectrum antibiotics in clinical use for treatment
of gram positive infections, particularly including critical care
antibiotics such as vancomycin, are limited in use and application
by their side effects of gastrointestinal upset and diarrhea and
the development of resistance, particularly in connection with
continued or long-term use.
[0011] It is evident from the deficiencies and problems associated
with current traditional antibacterial agents that there still
exists a need in the art for additional specific bacterial agents,
combinations and therapeutic modalities, particularly without high
risks of acquired resistance. Accordingly, there is a commercial
need for new antibacterial approaches, especially those that
operate via new modalities or provide new combinations to
effectively kill pathogenic bacteria.
[0012] The citation of references herein shall not be construed as
an admission that such is prior art to the present invention.
SUMMARY OF THE INVENTION
[0013] The present application relates to combinations of
bacteriophage lysin(s) with antibiotic for rapid and effective
killing of gram positive bacteria. In accordance with the
invention, the lysin PlySs2, which demonstrates broad killing
activity against multiple bacteria, particularly gram-positive
bacteria, including Staphylococcus and Streptococcus bacterial
strains, provides remarkable synergy in combination with
antibiotic(s) and can significantly reduce the effective MIC doses
required for antibiotic(s).
[0014] The lysin may be combined with broad spectrum gram positive
antibiotic(s), including one or more of vancomycin, daptomycin,
linezolid or oxacillin, including related or similar antibiotics.
In a particular aspect, PlySs2 lysin is combined with daptomycin to
provide synergistic killing activity against gram-positive
bacteria, including Staphylococci, particularly including MRSA. In
a particular aspect, PlySs2 lysin is combined with vancomycin to
provide synergistic killing activity against Staphylococci,
including MRSA. In a particular aspect, PlySs2 lysin is combined
with linezolid to provide synergistic killing activity against
Staphylococci, including MRSA. In an aspect of the invention,
combination with PlySs2 lysin significantly reduces the dose of
antibiotic required to kill a gram positive bacteria, such as S.
aureus.
[0015] In accordance with the invention, combinations of PlySs2
lysin and antibiotic, including antibiotic of distinct type or
class, particularly including daptomycin, vancomycin, linezolid or
oxacillin are effective to kill gram positive bacteria, including
S. aureus, at lower doses or with lower MIC than either alone. In
an aspect of the invention, lower dose formulations of lysin and of
antibiotic, including suitable for administration in combination or
separately simultaneously or in series, are provided wherein the
dose for effective killing or decolonization of a gram positive
infection are lower than the dose required if either are provided
alone. In particular, low dose formulations of antibiotic are
provided for administration in combination with lysin, particularly
PlySs2 lysin, administered simultaneously or in series, wherein the
dose for effective killing or decolonization of a gram positive
infection of the antibiotic are lower in combination with the lysin
than the dose required if antibiotic is provided or administered
alone.
[0016] In an aspect of the invention, lysins effective against
Staphylococci are combined with one or more of daptomycin,
vancomycin, linezolid or oxacillin, or related antibiotic
compounds, to kill gram positive bacteria, including S. aureus, at
lower doses or with lower MIC than either alone. In an aspect of
the invention, lysins effective against Staphylococci are combined
with daptomycin, or related antibiotic compounds, to kill gram
positive bacteria, including S. aureus, at lower doses or with
lower MIC than either alone. In an aspect of the invention, lysins
effective against Staphylococci are combined with one or more of
vancomycin, or related antibiotic compounds, to kill gram positive
bacteria, including S. aureus, at lower doses or with lower MIC
than either alone. In a particular aspect the antibiotic is
combined with PlySs2 lysin or a variant thereof. In an aspect of
the invention, the combination of lysin with daptomycin circumvents
the effect of surfactant to reduce daptomycin activity. In
combination with lysin, such as PlySs2 lysin, daptomycin is
rendered effective in killing S. aureus and in treating or
ameliorating bacteremia in an animal. Thus, in an aspect of the
invention, a method is provided for decolonization, inhibition or
treatment of a S. aureus infection in an animal comprising
administering to an animal a composition comprising or a
combination of PlySs2 lysin and daptomycin.
[0017] In accordance with the present invention, compositions and
methods comprising PlySs2 and one or more antibiotic are provided
for the prevention, disruption and treatment of bacterial infection
or colonization. In its broadest aspect, the present invention
provides use and application of a lysin having broad killing
activity against multiple bacteria, particularly gram-positive
bacteria, including Staphylococcus, Streptococcus, Enterococcus and
Listeria bacterial strains, in combination with antibiotic,
particularly in combination with daptomycin, vancomycin, linezolid
or oxacillin, or a related antibiotic, for the prevention,
amelioration or treatment of gram positive bacteria or gram
positive bacterial infections. The invention thus contemplates
treatment, decolonization, and/or decontamination of bacteria by
administration of or contact with a combination of PlySs2 lysin and
one or more antibiotic wherein one or more gram positive bacteria,
particularly one or more of Staphylococcus, Streptococcus,
Enterococcus and Listeria bacteria, is suspected or present. In one
such aspect, PlySs2 lysin is combined with daptomycin. In a further
aspect, PlySs2 lysin is combined with vancomycin. In another
aspect, PlySs2 lysin is combined with linezolid. In an additional
aspect, PlySs2 lysin is combined with oxacillin. In each instance
the antibiotic includes or encompasses related antibiotics,
including those of the same class or family or with similar or
related structures.
[0018] In accordance with the present invention, bacteriophage
lysin derived from Streptococcus suis bacteria are utilized in the
methods and compositions of the invention. The lysin polypeptide(s)
of use in the present invention, particularly PlySs2 lysin as
provided herein and in FIG. 29 (SEQ ID NO: 1), are unique in
demonstrating broad killing activity against multiple bacteria,
particularly gram-positive bacteria, including Staphylococcus,
Streptococcus, Enterococcus and Listeria bacterial strains. In one
such aspect, the PlySs2 lysin is capable of killing Staphylococcus
aureus strains and bacteria in combination with antibiotic,
particularly in combination with daptomycin, vancomycin, oxacillin
or linezolid, as demonstrated herein. PlySs2 is effective against
antibiotic-resistant Staphylococcus aureus such as
methicillin-resistant Staphylococcus aureus (MRSA), vancomycin
resistant Staphylococcus aureus (VRSA), daptomycin-resistant
Staphylococcus aureus (DRSA) and linezolid-resistant Staphylococcus
aureus (LRSA). PlySs2 is effective against vancomycin
intermediate-sensitivity Staphylococcus aureus (VISA).
[0019] In an aspect of the invention, a method is provided of
killing gram-positive bacteria comprising the step of contacting
the bacteria with a combination of PlySs2 lysin and one or more
antibiotic, the combination comprising an amount of an isolated
lysin polypeptide effective to kill gram-positive bacteria,
including S. aureus, the isolated lysin polypeptide comprising the
PlySs2 lysin polypeptide or variants thereof effective to kill
gram-positive bacteria, wherein the amount of PlySs2 required to be
effective to kill gram-positive bacteria, including S. aureus, in
the presence of antibiotic is significantly less than in the
absence of antibiotic. The isolated PlySs2 lysin polypeptide may
comprise the amino acid sequence provided in FIG. 29 (SEQ ID NO: 1)
or variants thereof having at least 80% identity, 85% identity, 90%
identity, 95% identity or 99% identity to the polypeptide of FIG.
29 (SEQ ID NO: 1) and effective to kill the gram-positive
bacteria.
[0020] In an aspect of the invention, a method is provided of
killing gram-positive bacteria comprising the step of contacting
the bacteria with a combination of PlySs2 lysin and one or more
antibiotic, the combination comprising an amount of an isolated
lysin polypeptide effective to kill gram-positive bacteria,
including S. aureus, the isolated lysin polypeptide comprising the
PlySs2 lysin polypeptide or variants thereof effective to kill
gram-positive bacteria, wherein the amount of antibiotic required
to be effective to kill gram-positive bacteria, including S.
aureus, in the presence of PlySs2 is significantly less than in the
absence of PlySs2.
[0021] As demonstrated in accordance with the present invention,
lysin as provided herein, particularly including lysin with
activity against Staphylococcus and Streptococcus bacteria,
particularly including PlySs2, acts synergistically with
antibiotics, particularly antibiotics of different class and
anti-bacterial mechanism. Thus, in accordance with the invention
PlySs2 lysins or active variants thereof demonstrate enhanced
activity in combination with antibiotics, including each of
antibiotics affecting cell wall synthesis such as glycopeptides,
penicillins which inhibit formation of peptidoglycan, protein
synthesis inhibitors, and lipopeptide antibiotic. In each instance
the antibacterial activity of both lysin and antibiotic is
significantly enhanced in combination. Combination with
glycopeptides antibiotic is evidenced by vancomycin, combination
with penicillin class is evidenced by oxacillin, combination with
protein synthesis inhibitor antibiotic including the class of
oxazolidinone is evidenced by linezolid, and combination with
lipopeptide antibiotic is evidenced by daptomycin. The present
invention includes and contemplates combinations and enhanced
activity with the demonstrated antibiotics as well as alternative
members of their class or a related antibiotic.
[0022] Thus, in an aspect of the invention, a method is provided of
killing gram-positive bacteria comprising the step of contacting
the bacteria with a combination of lysin and daptomycin or a
related antibiotic, the combination comprising an amount of an
isolated lysin polypeptide effective to kill gram-positive
bacteria, including S. aureus, wherein the amount of daptomycin or
related antibiotic required to be effective to kill gram-positive
bacteria, including S. aureus, in the presence of lysin is
significantly less than in the absence of lysin.
[0023] In a further aspect, a method is provided of killing
gram-positive bacteria comprising the step of contacting the
bacteria with a combination of lysin and vancomycin or a related
antibiotic, the combination comprising an amount of an isolated
lysin polypeptide effective to kill gram-positive bacteria,
including S. aureus, wherein the amount of vancomycin or related
antibiotic required to be effective to kill gram-positive bacteria,
including S. aureus, in the presence of lysin is significantly less
than in the absence of lysin.
[0024] In a further aspect, a method is provided of killing
gram-positive bacteria comprising the step of contacting the
bacteria with a combination of lysin and oxacillin or a related
antibiotic, the combination comprising an amount of an isolated
lysin polypeptide effective to kill gram-positive bacteria,
including S. aureus, wherein the amount of oxacillin or related
antibiotic required to be effective to kill gram-positive bacteria,
including S. aureus, in the presence of lysin is significantly less
than in the absence of lysin.
[0025] In a further aspect, a method is provided of killing
gram-positive bacteria comprising the step of contacting the
bacteria with a combination of lysin and linezolid or a related
antibiotic, the combination comprising an amount of an isolated
lysin polypeptide effective to kill gram-positive bacteria,
including S. aureus, wherein the amount of linezolid or related
antibiotic required to be effective to kill gram-positive bacteria,
including S. aureus, in the presence of lysin is significantly less
than in the absence of lysin.
[0026] The invention also provides a method of killing
antibiotic-resistant gram positive bacteria comprising contacting
the antibiotic-resistant bacteria with a lysin capable of killing
Staphylococcal bacteria. In one such aspect, the
antibiotic-resistant bacteria is contacted with lysin, particularly
PlySs2, in combination with an antibiotic to which the bacteria are
sensitive to, or in combination with antibiotic to which the
bacteria are resistant. In one such aspect of the method, the lysin
is PLySs2. In one such aspect, the lysin is a polypeptide
comprising the amino acid sequence provided in FIG. 29 (SEQ ID NO:
1) or variants thereof having at least 80% identity, 85% identity,
90% identity, 95% identity or 99% identity to the polypeptide of
FIG. 29 (SEQ ID NO: 1) and effective to kill gram-positive
bacteria, particularly S. aureus.
[0027] The invention provides such a method of killing daptomycin
resistant gram positive bacteria comprising contacting the
daptomycin resistant bacteria with a lysin capable of killing
Staphylococcal bacteria. Such method may include combination of the
lysin with daptomycin and/or with another antibiotic. In one such
aspect of the method, the lysin is PLySs2 as provided herein.
[0028] The invention further provides such a method of killing
vancomycin resistant gram positive bacteria comprising contacting
the vancomycin resistant bacteria with a lysin capable of killing
Staphylococcal bacteria. Such method may include combination of the
lysin with vancomycin and/or with another antibiotic. In one such
aspect of the method, the lysin is PLySs2.
[0029] In an aspect of the above methods, the methods are performed
in vitro, ex vivo, or along with implantation or placement of a
device in vivo so as to sterilize or decontaminate a solution,
material or device, particularly intended for use by or in a
human.
[0030] In a further aspect, a method is provided of enhancing
antibiotic effectiveness in killing or decolonizing gram-positive
bacteria comprising the step of contacting the bacteria with a
combination of lysin, particularly PlySs2, and one or more
antibiotic, wherein the amount of antibiotic required to be
effective to kill or decolonize the gram-positive bacteria,
including S. aureus, in the presence of lysin is significantly less
than in the absence of lysin. In one such aspect, a method is
providing for enhancing or facilitating the effectiveness of
daptomycin or a related antibiotic against Streptococcal pneumonia
comprising administering a lysin, particularly PlySs2, in
combination with daptomycin. In a particular such method or aspect,
daptomycin is effective against Streptococcal pneumonia when
administered in combination with or subsequent to administration of
lysin, particularly PlySs2, at a daptomycin dose which is
ineffective in the absence of lysin, particularly PlySs2.
[0031] The invention provides a method for reducing a population of
gram-positive bacteria comprising the step of contacting the
bacteria with a composition comprising an amount of an isolated
lysin polypeptide independently ineffective to kill the
gram-positive bacteria and an amount of antibiotic independently
ineffective to kill the gram-positive bacteria. The antibiotic may
be a glycopeptide, penicillin, protein synthesis inhibitor,
ozalidinone or lipopeptide. Such method may include an antibiotic
selected from vancomycin, daptomycin, linezolid and oxacillin. In
an aspect, the isolated lysin polypeptide comprises the amino acid
sequence of FIG. 29 or SEQ ID NO: 1) or variants thereof having at
least 80% identity to the polypeptide of FIG. 29 or SEQ ID NO: 1
and effective to kill the gram-positive bacteria.
[0032] The invention provides a method for reducing a population of
gram-positive bacteria comprising the step of contacting the
bacteria with a composition comprising an amount of an isolated
lysin polypeptide independently ineffective to kill the
gram-positive bacteria and an amount of daptomycin independently
ineffective to kill the gram-positive bacteria. In an aspect, the
isolated lysin polypeptide comprises the amino acid sequence of
FIG. 29 (SEQ ID NO: 1) or variants thereof having at least 80%
identity to the polypeptide of FIG. 29 (SEQ ID NO:1) and effective
to kill the gram-positive bacteria.
[0033] In any such above method or methods, the susceptible,
killed, dispersed or treated bacteria may be selected from
Staphylococcus aureus, Listeria monocytogenes, Staphylococcus
simulans, Streptococcus suis, Staphylococcus epidermidis,
Streptococcus equi, Streptococcus equi zoo, Streptococcus
agalactiae (GBS), Streptococcus pyogenes (GAS), Streptococcus
sanguinis, Streptococcus gordonii, Streptococcus dysgalactiae,
Group G Streptococcus, Group E Streptococcus, Enterococcus faecalis
and Streptococcus pneumonia.
[0034] In accordance with any of the methods of the invention, the
susceptible bacteria may be an antibiotic resistant bacteria. The
bacteria may be methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin intermediate-sensitivity Staphylococcus aureus (VISA),
vancomycin resistant Staphylococcus aureus (VRSA),
daptomycin-resistant Staphylococcus aureus (DRSA), or
linezolid-resistant Staphylococcus aureus (LRSA). The susceptible
bacteria may be a clinically relevant or pathogenic bacteria,
particularly for humans. In an aspect of the method(s), the lysin
polypeptide(s) is effective to kill Staphylococcus, Streptococcus,
Enterococcus and Listeria bacterial strains.
[0035] In an additional aspect or embodiment of the methods and
compositions provided herein, another distinct staphylococcal
specific lysin is used herein alone or in combination with the
PlySs2 lysin as provided and described herein. In one such aspect
or embodiment of the methods and compositions provided herein, the
staphylococcal specific lysin ClyS is used herein alone or in
combination with the PlySs2 lysin as provided and described
herein.
[0036] The invention provides methods for enhancing or facilitating
antibiotic activity comprising administering a combination together
or in series of lysin, particularly PlySs2 lysin, and one or more
antibiotic. In an aspect thereof, antibiotic activity is enhanced
or facilitated by at least 10 fold, at least 16 fold, at least 20
fold, at least 24 fold, at least 30 fold, at least 40 fold, at
least 50 fold, at least 70 fold, at least 80 fold at least 100
fold, more than 10 fold, more than 20 fold, more than 50 fold, more
than 100 fold. The invention provides methods for enhancing or
facilitating lysin activity, particularly PlySs2 lysin, comprising
administering a combination together or in series of lysin,
particularly PlySs2 lysin, and one or more antibiotic. In an aspect
thereof, the activity of lysin, particularly PlySs2 is enhanced at
least 2 fold, at least 4 fold, at least 8 fold, at least 10 fold,
up to 10 fold, up to 16 fold, up to 20 fold.
[0037] The invention includes a method of potentiating antibiotic
activity against gram-positive bacteria in biological fluids having
surfactant-like activity comprising administering antibiotic in
combination with PlySs2 lysin comprising the amino acid sequence
provided in FIG. 29 (SEQ ID NO: 1) or variants thereof having at
least 80% identity, 85% identity, 90% identity, 95% identity or 99%
identity to the polypeptide of FIG. 29 (SEQ ID NO: 1) and effective
to kill gram-positive bacteria, wherein the antibiotic is effective
in combination with PlySs2 at doses that the antibiotic is
ineffective in the absence of PlySs2. In an aspect of the method,
the antibiotic is daptomycin or a related compound. In an aspect,
the bacteria is S. pneumoniae.
[0038] Other objects and advantages will become apparent to those
skilled in the art from a review of the following description which
proceeds with reference to the following illustrative drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 depicts time kill curves of various MRSA strains in
the presence of added daptomycin, vancomycin or PlySs2 lysin.
[0040] FIG. 2 depicts time kill curves of various MSSA strains in
the presence of added daptomycin, vancomycin, oxacillin or PlySs2
lysin.
[0041] FIG. 3 provides a summary plot of time kill curves of
various MRSA and MSSA strains in the presence of added daptomycin,
vancomycin or PlySs2 lysin.
[0042] FIG. 4 provides composite time kill curves of PlySS2 and
antibiotics on S. aureus cells in vitro. (A, B) Composite time-kill
curves of PlySS2 compared to oxacillin (OXA), vancomycin (VAN), and
daptomycin (DAP) against sets of 20 MSSA and 42 MRSA strains,
respectively. In each individual analysis, drug concentrations
correspond to strain-specific 1.times.MIC values. Mean values
(.+-.standard error of the mean) are shown for each time-point. (C,
D) Titration analysis of PlySS2 against sets of 15 contemporary
clinical MSSA and MRSA isolates, respectively. In each individual
analysis, PlySS2 concentrations correspond to strain-specific MIC
values. 4.lamda., 1.times., and 0.25.lamda. MIC concentrations were
used. (E,F) Transmission electron micrographs (3300.times.
magnification) of S. aureus cells (strain MW2) before and after 3
second treatment with 8 g/mL PlySS2. Scale bars correspond to 2
.mu.M. Lysis results in the loss of darkly stained cytoplasmic
components.
[0043] FIG. 5 shows time kill curves for MRSA strains treated with
PlySs2 and vancomycin alone or in combination at the noted sub MIC
doses.
[0044] FIG. 6 shows time kill curves for MRSA strains treated with
PlySs2 and daptomycin alone or in combination at the noted sub MIC
doses.
[0045] FIG. 7 depicts time kill curves for MRSA strain 650 (O52C
Tomasz) in the presence of added daptomycin and PlySs2 lysin alone
or in combination at the noted MIC or dose.
[0046] FIG. 8A-8F shows that PlySs2 synergizes with antibiotics
across multiple strains in-vitro and depicts time-kill results for
MSSA strains treated with PlySs2 and oxacillin (A,B); MRSA strains
treated with Plyss2 and vancomycin (C,D), MRSA strains treated with
PlySs2 and daptomycin (E,F). In panels A, C, and E time-kill data
are shown for three individual strains, MSSA strain JMI 7140, MRSA
strain JMI 3340 and MRSA strain JMI 3345 respectively. (A) Values
are shown for growth, growth control (no PlySs2 or antibiotic),
PlySs2 0.13.times.MIC, oxacillin (OXA) 0.5.times.MIC, PlySS2+Oxa
combination of indicated drug concentrations. (C) Values are shown
for growth, growth control (no PlySs2 or antibiotic), PlySs2
0.13.times.MIC, vancomycin (VAN) 0.5.times.MIC, PlySS2+VAN
combination of indicated drug concentrations. (E) Values are shown
for growth, growth control (no PlySs2 or antibiotic), PlySs2
0.25.times.MIC, daptomycin (DAP) 0.5.times.MIC, PlySS2+DAP
combination of indicated drug concentrations. In panels B, D, and F
the log change in cfu/ml between the combination-treated culture
and the untreated growth control over 6 hours are shown for
collections of strains. The horizontal dotted lines indicate the 2
log cutoff required for scoring time-kill synergy. Decreases in
log.sub.10 colony counts (or .DELTA. Log.sub.10 CFU/mL) are shown
for cultures treated for 6 hours with drug combination, compared to
cultures treated with the most active single agent. Synergy is
defined by the CLSI as a .gtoreq.2-log.sub.10 decrease in CFU/mL
and is denoted in the figure by the dashed line. Key: .DELTA. Log
10 CFU/mL=change in log.sub.10 colony-forming units.
[0047] FIG. 9 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
553 in the presence of reducing agent (BME).
[0048] FIG. 10 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
553 in the absence of reducing agent (BME).
[0049] FIG. 11 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
223 in the presence of BME.
[0050] FIG. 12 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
223 in the absence of BME.
[0051] FIG. 13 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
270 in the presence and absence of BME.
[0052] FIG. 14 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
269 in the presence and absence of BME.
[0053] FIG. 15 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
241 in the presence and absence of BME.
[0054] FIG. 16 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
263 in the presence and absence of BME.
[0055] FIG. 17 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
650 in the presence and absence of BME.
[0056] FIG. 18 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
828 in the presence and absence of BME.
[0057] FIG. 19 depicts representative isobolograms depicting FIC
values of lysin PlySs2 versus FIC values of antibiotic. PlySs2
versus antibiotics oxacillin, vancomycin and daptomycin are
depicted against MSSA strains and MRSA strains as noted. Oxacillin
and PlySs2 are evaluated versus MSSA strain JMI 33611. PlySs2 and
vancomycin are evaluated versus MSSA strain JMI 9365 and MRSA
strain JMI 6456. Daptomycin and PlySs2 are evaluated versus MSSA
strain JMI 33611 and MRSA JMI 3345.
[0058] FIG. 20 provides a time course of S. aureus staining by
BODIPY-labeled daptomycin (A) and vancomycin (B) in the absence and
presence of sub-MIC amounts of PlySs2.
[0059] FIG. 21 depicts the fold change in MIC value against MRSA
strain MW2 and MSSA strain ATCC 29213 treated with PlySs2 or
daptomycin in the presence of varying amounts of surfactant (from
1.25 to 15% surfactant).
[0060] FIG. 22 provides a panel of dose dilutions of pairings of
daptomycin and PlySs2 at the noted concentrations on MRSA strain
269 in the presence of 15% surfactant (Survanta).
[0061] FIG. 23 provides a compiled graph of % survival of mice (50
animals) challenged with MRSA strain 269 (MW2) in several
experiments having bacterial inoculum strengths of
1.1-3.1.times.10.sup.6 CFU and treated with daptomycin or PlySs2
alone or in combination.
[0062] FIG. 24 depicts % survival of mice challenged with MRSA
strain 220 at 2.65.times.10.sup.6 CFU and treated with the
indicated doses of daptomycin or PlySs2 alone or in
combination.
[0063] FIG. 25 depicts % survival of mice challenged with MRSA
strain 833 at 1.4.times.10.sup.6 CFU and treated with the indicated
doses of daptomycin or PlySs2 alone or in combination.
[0064] FIG. 26 depicts % survival of mice challenged with MRSA
strain 833 at 2.0.times.10.sup.6 CFU and treated with the indicated
doses of daptomycin or PlySs2 alone or in combination.
[0065] FIG. 27 depicts survival curves of combination therapy
compared to mono-therapies in murine models of bacteremia. Mice
were challenged with either 7.5.times.10.sup.6 cfu/mouse i.p. (low
challenge model--panel a) or 10.sup.9 cfu/mouse i.p. (high
challenge model--panels b-f) at time 0 and were treated with either
antibiotic, PlySs2, combination of PlySs2 and antibiotic, or
control and the resulting survival data are shown in Kaplan-Meier
format. All doses were administered as a single bolus dose except
for vancomycin (BID, panel e) and oxacillin (QID, panel f) which
were administered as multiple doses over the first 24 hr period.
Routes of administration were PlySs2 (i.p.), daptomycin and
vancomycin (subcutaneous), and oxacillin (intramuscular). P values
were calculated for the combinations versus antibiotic alone. (A)
Low challenge model using MRSA strain MW2 with daptomycin at 2
mg/kg and PlySs2 at 1.25 mg/kg. Dosing at 4 hr post inoculation,
n=30, P<0.0001. (B) High challenge model using MRSA strain MW2
with daptomycin at 50 mg/kg and PlySs2 at 5.25 mg/kg. Dosing at 2
hr post inoculation, n=45, P<0.0001. (C) same as B using MRSA
strain 738, n=30, P<0.0001. (D) same as B using MRSA strain 832,
n=30, P<0.0001. (E) High challenge model using MRSA strain MW2
with vancomycin at 110 mg/kg BID and PlySs2 at 5.25 mg/kg. Dosing
initiated at 2 hr post-inoculation, n=30, P<0.0001. (F) High
challenge model using MSSA strain ATCC 25923 with oxacillin at 200
mg/kg QID and PlySs2 at 5.25 mg/kg. Dosing initiated at 2 hr
post-inoculation, n=30 P<0.0001.
[0066] FIG. 28 depicts MIC of daptomycin and PlySs2 on an MSSA and
a MRSA strain with passage number and development of daptomycin
resistance. PlySs2 MIC drops showing PlySs2 increased sensitivity
with increased daptomycin resistance.
[0067] FIG. 29 provides the amino acid sequence (SEQ ID NO: 1) and
encoding nucleic acid sequence (SEQ ID NO: 2) of the lysin PlySs2.
The N-terminal CHAP domain and the C-terminal SH-3 domain of the
PlySs2 lysin are shaded, with the CHAP domain (SEQ ID NO: 3)
starting with LNN . . . and ending with . . . YIT and the SH-3
domain (SEQ ID NO: 4) starting with RSY . . . and ending with . . .
VAT. The CHAP domain active-site residues (Cys.sub.26, His.sub.102,
Glu.sub.118, and Asn.sub.120) identified by homology to PDB 2K3A
(Rossi P et al (2009) Proteins 74:515-519) are underlined.
[0068] FIG. 30 depicts fold change in daptomycin MIC value as a
function of days of serial passage under resistance selection
conditions in the presence of daptomycin alone or daptomycin with
sub-MIC amounts of PlySs2 lysin for multiple cultures (three
independent cultures of each).
[0069] FIG. 31 depicts fold change in vancomycin MIC value as a
function of days of serial passage under resistance selection
conditions in the presence of daptomycin alone or daptomycin with
sub-MIC amounts PlySs2 lysin for multiple cultures (three
independent cultures of each).
DETAILED DESCRIPTION
[0070] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. 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.
M., 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).
[0071] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0072] The terms "PlySs lysin(s)", "PlySs2 lysin", "PlySs2" and any
variants not specifically listed, may be used herein
interchangeably, and as used throughout the present application and
claims refer to proteinaceous material including single or multiple
proteins, and extends to those proteins having the amino acid
sequence data described herein and presented in FIG. 29 and SEQ ID
NO: 1, and the profile of activities set forth herein and in the
Claims. Accordingly, proteins displaying substantially equivalent
or altered activity are likewise contemplated. These modifications
may be deliberate, for example, such as modifications obtained
through site-directed mutagenesis, or may be accidental, such as
those obtained through mutations in hosts that are producers of the
complex or its named subunits. Also, the terms "PlySs lysin(s)",
"PlySs2 lysin", "PlySs2" are intended to include within their scope
proteins specifically recited herein as well as all substantially
homologous analogs, fragments or truncations, and allelic
variations. PlySs2 lysin is described in U.S. Patent Application
61/477,836 and PCT Application PCT/US2012/34456. A more recent
paper Gilmer et al describes PlySs2 lysin (Gilmer D B et al (2013)
Antimicrob Agents Chemother Epub 2013 April 9 [PMID 23571534]).
[0073] The term "ClyS", "ClyS lysin" refers to a chimeric lysin
ClyS, with activity against Staphylococci bacteria, including
Staphylococcus aureus, is detailed in WO 2010/002959 and also
described in Daniel et al (Daniel, A et al (2010) Antimicrobial
Agents and Chemother 54(4):1603-1612). Exemplary ClyS amino acid
sequence is provided in SEQ ID NO: 5.
[0074] A "lytic enzyme" includes any bacterial cell wall lytic
enzyme that kills one or more bacteria under suitable conditions
and during a relevant time period. Examples of lytic enzymes
include, without limitation, various amidase cell wall lytic
enzymes. In a particular aspect, a lytic enzyme refers to a
bacteriophage lytic enzyme. A "bacteriophage lytic enzyme" refers
to a lytic enzyme extracted or isolated from a bacteriophage or a
synthesized lytic enzyme with a similar protein structure that
maintains a lytic enzyme functionality.
[0075] A lytic enzyme is capable of specifically cleaving bonds
that are present in the peptidoglycan of bacterial cells to disrupt
the bacterial cell wall. It is also currently postulated that the
bacterial cell wall peptidoglycan is highly conserved among most
bacteria, and cleavage of only a few bonds to may disrupt the
bacterial cell wall. Examples of lytic enzymes that cleave these
bonds are muramidases, glucosaminidases, endopeptidases, or
N-acetyl-muramoyl-L-alanine amidases. Fischetti et al (1974)
reported that the C1 streptococcal phage lysin enzyme was an
amidase. Garcia et al (1987, 1990) reported that the Cp1 lysin from
a S. pneumoniae from a Cp-1 phage was a lysozyme. Caldentey and
Bamford (1992) reported that a lytic enzyme from the phi 6
Pseudomonas phage was an endopeptidase, splitting the peptide
bridge formed by melo-diaminopimilic acid and D-alanine. The E.
coli T1 and T6 phage lytic enzymes are amidases as is the lytic
enzyme from Listeria phage (ply) (Loessner et al, 1996). There are
also other lytic enzymes known in the art that are capable of
cleaving a bacterial cell wall.
[0076] A "lytic enzyme genetically coded for by a bacteriophage"
includes a polypeptide capable of killing a host bacteria, for
instance by having at least some cell wall lytic activity against
the host bacteria. The polypeptide may have a sequence that
encompasses native sequence lytic enzyme and variants thereof. The
polypeptide may be isolated from a variety of sources, such as from
a bacteriophage ("phage"), or prepared by recombinant or synthetic
methods. The polypeptide may, for example, comprise a
choline-binding portion at the carboxyl terminal side and may be
characterized by an enzyme activity capable of cleaving cell wall
peptidoglycan (such as amidase activity to act on amide bonds in
the peptidoglycan) at the amino terminal side. Lytic enzymes have
been described which include multiple enzyme activities, for
example two enzymatic domains, such as PlyGBS lysin. Further, other
lytic enzymes have been described containing only a catalytic
domain and no cell wall binding domain.
[0077] "A native sequence phage associated lytic enzyme" includes a
polypeptide having the same amino acid sequence as an enzyme
derived from a bacterial genome (i.e., a prophage). Such native
sequence enzyme can be isolated or can be produced by recombinant
or synthetic means.
[0078] The term "native sequence enzyme" encompasses naturally
occurring forms (e.g., alternatively spliced or altered forms) and
naturally-occurring variants of the enzyme. In one embodiment of
the invention, the native sequence enzyme is a mature or
full-length polypeptide that is genetically coded for by a gene
from a bacteriophage specific for Streptococcus suis. Of course, a
number of variants are possible and known, as acknowledged in
publications such as Lopez et al., Microbial Drug Resistance 3:
199-211 (1997); Garcia et al., Gene 86: 81-88 (1990); Garcia et
al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al.,
Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al.,
Streptococcal Genetics (J. J. Ferretti and Curtis eds., 1987);
Lopez et al., 1-BMS Microbiol. Lett. 100: 439-448 (1992); Romero et
al., J. Bacteriol. 172: 5064-5070 (1990); Ronda et al., Eur. J.
Biochem. 164: 621-624 (1987) and Sanchez et al., Gene 61: 13-19
(1987). The contents of each of these references, particularly the
sequence listings and associated text that compares the sequences,
including statements about sequence homologies, are specifically
incorporated by reference in their entireties.
[0079] "A variant sequence lytic enzyme" includes a lytic enzyme
characterized by a polypeptide sequence that is different from that
of a lytic enzyme, but retains functional activity. The lytic
enzyme can, in some embodiments, be genetically coded for by a
bacteriophage specific for Streptococcus suis as in the case of
PlySs2 having a particular amino acid sequence identity with the
lytic enzyme sequence(s) hereof, as provided in FIG. 29 and in SEQ
ID NO: 1. For example, in some embodiments, a functionally active
lytic enzyme can kill Streptococcus suis bacteria, and other
susceptible bacteria as provided herein, including as shown in
TABLE 1, 2 and 3, by disrupting the cellular wall of the bacteria.
An active lytic enzyme may have a 60, 65, 70, 75, 80, 85, 90, 95,
97, 98, 99 or 99.5% amino acid sequence identity with the lytic
enzyme sequence(s) hereof, as provided in FIG. 29 (SEQ ID NO: 1,
SEQ ID NO: 3 or SEQ ID NO: 4). Such phage associated lytic enzyme
variants include, for instance, lytic enzyme polypeptides wherein
one or more amino acid residues are added, or deleted at the N or C
terminus of the sequence of the lytic enzyme sequence(s) hereof, as
provided in FIG. 29 (SEQ ID NO: 1).
[0080] In a particular aspect, a phage associated lytic enzyme will
have at least about 80% or 85% amino acid sequence identity with
native phage associated lytic enzyme sequences, particularly at
least about 90% (e.g. 90%) amino acid sequence identity. Most
particularly a phage associated lytic enzyme variant will have at
least about 95% (e.g. 95%) amino acid sequence identity with the
native phage associated the lytic enzyme sequence(s) hereof, as
provided in FIG. 29 (SEQ ID NO: 1) for PlySs2 lysin, or as
previously described for ClyS including in WO 2010/002959 and also
described in Daniel et al (Daniel, A et al (2010) Antimicrobial
Agents and Chemother 54(4):1603-1612) and SEQ ID NO: 5.
[0081] "Percent amino acid sequence identity" with respect to the
phage associated lytic enzyme sequences identified is defined
herein as the percentage of amino acid residues in a candidate
sequence that are identical with the amino acid residues in the
phage associated lytic enzyme sequence, after aligning the
sequences in the same reading frame and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity.
[0082] "Percent nucleic acid sequence identity" with respect to the
phage associated lytic enzyme sequences identified herein is
defined as the percentage of nucleotides in a candidate sequence
that are identical with the nucleotides in the phage associated
lytic enzyme sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity.
[0083] To determine the percent identity of two nucleotide or amino
acid sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps may be introduced in the sequence of a first
nucleotide sequence). The nucleotides or amino acids at
corresponding nucleotide or amino acid positions are then compared.
When a position in the first sequence is occupied by the same
nucleotide or amino acid as the corresponding position in the
second sequence, then the molecules are identical at that position.
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positionsX100).
[0084] The determination of percent identity between two sequences
may be accomplished using a mathematical algorithm. A non-limiting
example of a mathematical algorithm utilized for the comparison of
two sequences is the algorithm of Karlin et al., Proc. Natl. Acad.
Sci. USA, 90:5873-5877 (1993), which is incorporated into the
NBLAST program which may be used to identify sequences having the
desired identity to nucleotide sequences of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST may
be utilized as described in Altschul et al., Nucleic Acids Res,
25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
NBLAST) may be used. See the programs provided by National Center
for Biotechnology Information, National Library of Medicine,
National Institutes of Health.
[0085] "Polypeptide" includes a polymer molecule comprised of
multiple amino acids joined in a linear manner A polypeptide can,
in some embodiments, correspond to molecules encoded by a
polynucleotide sequence which is naturally occurring. The
polypeptide may include conservative substitutions where the
naturally occurring amino acid is replaced by one having similar
properties, where such conservative substitutions do not alter the
function of the polypeptide.
[0086] The term "altered lytic enzymes" includes shuffled and/or
chimeric lytic enzymes.
[0087] Phage lytic enzymes specific for bacteria infected with a
specific phage have been found to effectively and efficiently break
down the cell wall of the bacterium in question. The lytic enzyme
is believed to lack proteolytic enzymatic activity and is therefore
non-destructive to mammalian proteins and tissues when present
during the digestion of the bacterial cell wall. Furthermore,
because it has been found that the action of phage lytic enzymes,
unlike antibiotics, was rather specific for the target pathogen(s),
it is likely that the normal flora will remain essentially intact
(M. J. Loessner, G. Wendlinger, S. Scherer, Mol Microbiol 16,
1231-41. (1995) incorporated herein by reference). In fact, the
PlySs2 lysin, while demonstrating uniquely broad bacterial species
and strain killing, is comparatively and particularly inactive
against bacteria comprising the normal flora, including E. coli, as
described herein.
[0088] A lytic enzyme or polypeptide of use in the invention may be
produced by the bacterial organism after being infected with a
particular bacteriophage or may be produced or prepared
recombinantly or synthetically as either a prophylactic treatment
for preventing those who have been exposed to others who have the
symptoms of an infection from getting sick, or as a therapeutic
treatment for those who have already become ill from the infection.
In as much the lysin polypeptide sequences and nucleic acids
encoding the lysin polypeptides are described and referenced to
herein, the lytic enzyme(s)/polypeptide(s) may be preferably
produced via the isolated gene for the lytic enzyme from the phage
genome, putting the gene into a transfer vector, and cloning said
transfer vector into an expression system, using standard methods
of the art, including as exemplified herein. The lytic enzyme(s) or
polypeptide(s) may be truncated, chimeric, shuffled or "natural,"
and may be in combination. Relevant U.S. Pat. No. 5,604,109 is
incorporated herein in its entirety by reference. An "altered"
lytic enzyme can be produced in a number of ways. In a preferred
embodiment, a gene for the altered lytic enzyme from the phage
genome is put into a transfer or movable vector, preferably a
plasmid, and the plasmid is cloned into an expression vector or
expression system. The expression vector for producing a lysin
polypeptide or enzyme of the invention may be suitable for E. coli,
Bacillus, or a number of other suitable bacteria. The vector system
may also be a cell free expression system. All of these methods of
expressing a gene or set of genes are known in the art. The lytic
enzyme may also be created by infecting Streptococcus suis with a
bacteriophage specific for Streptococcus suis, wherein said at
least one lytic enzyme exclusively lyses the cell wall of said
Streptococcus suis having at most minimal effects on other, for
example natural or commensal, bacterial flora present.
[0089] A "chimeric protein" or "fusion protein" comprises all or
(preferably a biologically active) part of a polypeptide of use in
the invention operably linked to a heterologous polypeptide.
Chimeric proteins or peptides are produced, for example, by
combining two or more proteins having two or more active sites.
Chimeric protein and peptides can act independently on the same or
different molecules, and hence have a potential to treat two or
more different bacterial infections at the same time. Chimeric
proteins and peptides also may be used to treat a bacterial
infection by cleaving the cell wall in more than one location, thus
potentially providing more rapid or effective (or synergistic)
killing from a single lysin molecule or chimeric peptide.
[0090] A "heterologous" region of a DNA construct or peptide
construct is an identifiable segment of DNA within a larger DNA
molecule or peptide within a larger peptide molecule that is not
found in association with the larger molecule in nature. Thus, when
the heterologous region encodes a mammalian gene, the gene will
usually be flanked by DNA that does not flank the mammalian genomic
DNA in the genome of the source organism. Another example of a
heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences
having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a
heterologous region of DNA or peptide as defined herein.
[0091] The term "operably linked" means that the polypeptide of the
disclosure and the heterologous polypeptide are fused in-frame. The
heterologous polypeptide can be fused to the N-terminus or
C-terminus of the polypeptide of the disclosure. Chimeric proteins
are produced enzymatically by chemical synthesis, or by recombinant
DNA technology. A number of chimeric lytic enzymes have been
produced and studied. One example of a useful fusion protein is a
GST fusion protein in which the polypeptide of the disclosure is
fused to the C-terminus of a GST sequence. Such a chimeric protein
can facilitate the purification of a recombinant polypeptide of the
disclosure.
[0092] In another embodiment, the chimeric protein or peptide
contains a heterologous signal sequence at its N-terminus. For
example, the native signal sequence of a polypeptide of the
disclosure can be removed and replaced with a signal sequence from
another known protein.
[0093] The fusion protein may combine a lysin polypeptide with a
protein or polypeptide of having a different capability, or
providing an additional capability or added character to the lysin
polypeptide. The fusion protein may be an immunoglobulin fusion
protein in which all or part of a polypeptide of the disclosure is
fused to sequences derived from a member of the immunoglobulin
protein family. The immunoglobulin may be an antibody, for example
an antibody directed to a surface protein or epitope of a
susceptible or target bacteria. The immunoglobulin fusion protein
can alter bioavailability of a cognate ligand of a polypeptide of
the disclosure. Inhibition of ligand/receptor interaction may be
useful therapeutically, both for treating bacterial-associated
diseases and disorders for modulating (i.e. promoting or
inhibiting) cell survival. The fusion protein may include a means
to direct or target the lysin, including to particular tissues or
organs or to surfaces such as devices, plastic, membranes. Chimeric
and fusion proteins and peptides of the disclosure can be produced
by standard recombinant DNA techniques.
[0094] A modified or altered form of the protein or peptides and
peptide fragments, as disclosed herein, includes protein or
peptides and peptide fragments that are chemically synthesized or
prepared by recombinant DNA techniques, or both. These techniques
include, for example, chimerization and shuffling. As used herein,
shuffled proteins or peptides, gene products, or peptides for more
than one related phage protein or protein peptide fragments have
been randomly cleaved and reassembled into a more active or
specific protein. Shuffled oligonucleotides, peptides or peptide
fragment molecules are selected or screened to identify a molecule
having a desired functional property. Shuffling can be used to
create a protein that is more active, for instance up to 10 to 100
fold more active than the template protein. The template protein is
selected among different varieties of lysin proteins. The shuffled
protein or peptides constitute, for example, one or more binding
domains and one or more catalytic domains. When the protein or
peptide is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals, i.e.,
it is separated from chemical precursors or other chemicals which
are involved in the synthesis of the protein. Accordingly such
preparations of the protein have less than about 30%, 20%, 10%, 5%
(by dry weight) of chemical precursors or compounds other than the
polypeptide of interest.
[0095] The present invention also pertains to other variants of the
polypeptides useful in the invention. Such variants may have an
altered amino acid sequence which can function as either agonists
(mimetics) or as antagonists. Variants can be generated by
mutagenesis, i.e., discrete point mutation or truncation. An
agonist can retain substantially the same, or a subset, of the
biological activities of the naturally occurring form of the
protein. An antagonist of a protein can inhibit one or more of the
activities of the naturally occurring form of the protein by, for
example, competitively binding to a downstream or upstream member
of a cellular signaling cascade which includes the protein of
interest. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein can have fewer side
effects in a subject relative to treatment with the naturally
occurring form of the protein. Variants of a protein of use in the
disclosure which function as either agonists (mimetics) or as
antagonists can be identified by screening combinatorial libraries
of mutants, such as truncation mutants, of the protein of the
disclosure. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. There are a variety of
methods which can be used to produce libraries of potential
variants of the polypeptides of the disclosure from a degenerate
oligonucleotide sequence. Libraries of fragments of the coding
sequence of a polypeptide of the disclosure can be used to generate
a variegated population of polypeptides for screening and
subsequent selection of variants, active fragments or truncations.
Several techniques are known in the art for screening gene products
of combinatorial libraries made by point mutations or truncation,
and for screening cDNA libraries for gene products having a
selected property. The most widely used techniques, which are
amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. In this context, the smallest portion of a protein
(or nucleic acid that encodes the protein) according to embodiments
is an epitope that is recognizable as specific for the phage that
makes the lysin protein. Accordingly, the smallest polypeptide (and
associated nucleic acid that encodes the polypeptide) that can be
expected to bind a target or receptor, such as an antibody, and is
useful for some embodiments may be 8, 9, 10, 11, 12, 13, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, or 100 amino acids
long. Although small sequences as short as 8, 9, 10, 11, 12 or 15
amino acids long reliably comprise enough structure to act as
targets or epitopes, shorter sequences of 5, 6, or 7 amino acids
long can exhibit target or epitopic structure in some conditions
and have value in an embodiment. Thus, the smallest portion of the
protein(s) or lysin polypeptides provided herein, including as set
out in FIG. 29 (SEQ ID NO: 1) includes polypeptides as small as 5,
6, 7, 8, 9, 10, 12, 14 or 16 amino acids long.
[0096] Biologically active portions of a protein or peptide
fragment of the embodiments, as described herein, include
polypeptides comprising amino acid sequences sufficiently identical
to or derived from the amino acid sequence of the lysin protein of
the disclosure, which include fewer amino acids than the full
length protein of the lysin protein and exhibit at least one
activity of the corresponding full-length protein. Typically,
biologically active portions comprise a domain or motif with at
least one activity of the corresponding protein. An exemplary
domain sequence for the N terminal CHAP domain of the lysin of the
present invention is provided in FIG. 29 and SEQ ID NO: 3. An
exemplary domain sequence for the C terminal SH3 domain of the
lysin of the present invention is provided in FIG. 29 and SEQ ID
NO: 4. A biologically active portion of a protein or protein
fragment of the disclosure can be a polypeptide which is, for
example, 10, 25, 50, 100 less or more amino acids in length.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, or added can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of the native form of a polypeptide of the
embodiments.
[0097] Homologous proteins and nucleic acids can be prepared that
share functionality with such small proteins and/or nucleic acids
(or protein and/or nucleic acid regions of larger molecules) as
will be appreciated by a skilled artisan. Such small molecules and
short regions of larger molecules that may be homologous
specifically are intended as embodiments. Preferably the homology
of such valuable regions is at least 50%, 65%, 75%, 80%, 85%, and
preferably at least 90%, 95%, 97%, 98%, or at least 99% compared to
the lysin polypeptides provided herein, including as set out in
FIG. 29. These percent homology values do not include alterations
due to conservative amino acid substitutions.
[0098] Two amino acid sequences are "substantially homologous" when
at least about 70% of the amino acid residues (preferably at least
about 80%, at least about 85%, and preferably at least about 90 or
95%) are identical, or represent conservative substitutions. The
sequences of comparable lysins, such as comparable PlySs2 lysins,
or comparable ClyS lysins, are substantially homologous when one or
more, or several, or up to 10%, or up to 15%, or up to 20% of the
amino acids of the lysin polypeptide are substituted with a similar
or conservative amino acid substitution, and wherein the comparable
lysins have the profile of activities, anti-bacterial effects,
and/or bacterial specificities of a lysin, such as the PlySs2 lysin
and/or ClyS lysin, disclosed herein.
[0099] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the desired fuctional property of immunoglobulin-binding is
retained by the polypeptide. NH.sub.2 refers to the free amino
group present at the amino terminus of a polypeptide. COOH refers
to the free carboxy group present at the carboxy terminus of a
polypeptide. In keeping with standard polypeptide nomenclature, J.
Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid
residues are shown in the following Table of Correspondence:
TABLE-US-00001 TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter
AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met
methionine A Ala alanine S Ser serine I Ile isoleucine L Leu
leucine T Thr threonine V Val valine P Pro proline K Lys lysine H
His histidine Q Gln glutamine E Glu glutamic acid W Trp tryptophan
R Arg arginine D Asp aspartic acid N Asn asparagine C Cys
cysteine
[0100] Mutations can be made in the amino acid sequences, or in the
nucleic acid sequences encoding the polypeptides and lysins herein,
including in the lysin sequences set out in FIG. 29 (SEQ ID NO: 1),
or in active fragments or truncations thereof, such that a
particular codon is changed to a codon which codes for a different
amino acid, an amino acid is substituted for another amino acid, or
one or more amino acids are deleted. Such a mutation is generally
made by making the fewest amino acid or nucleotide changes
possible. A substitution mutation of this sort can be made to
change an amino acid in the resulting protein in a non-conservative
manner (for example, by changing the codon from an amino acid
belonging to a grouping of amino acids having a particular size or
characteristic to an amino acid belonging to another grouping) or
in a conservative manner (for example, by changing the codon from
an amino acid belonging to a grouping of amino acids having a
particular size or characteristic to an amino acid belonging to the
same grouping). Such a conservative change generally leads to less
change in the structure and function of the resulting protein. A
non-conservative change is more likely to alter the structure,
activity or function of the resulting protein. The present
invention should be considered to include sequences containing
conservative changes which do not significantly alter the activity
or binding characteristics of the resulting protein.
[0101] Thus, one of skill in the art, based on a review of the
sequence of the PlySs2 lysin polypeptide provided herein and on
their knowledge and the public information available for other
lysin polypeptides, can make amino acid changes or substitutions in
the lysin polypeptide sequence. Amino acid changes can be made to
replace or substitute one or more, one or a few, one or several,
one to five, one to ten, or such other number of amino acids in the
sequence of the lysin(s) provided herein to generate mutants or
variants thereof. Such mutants or variants thereof may be predicted
for function or tested for function or capability for killing
bacteria, including Staphylococcal, Streptococcal, Listeria, or
Enterococcal bacteria, and/or for having comparable activity to the
lysin(s) as described and particularly provided herein. Thus,
changes can be made to the sequence of lysin, and mutants or
variants having a change in sequence can be tested using the assays
and methods described and exemplified herein, including in the
examples. One of skill in the art, on the basis of the domain
structure of the lysin(s) hereof can predict one or more, one or
several amino acids suitable for substitution or replacement and/or
one or more amino acids which are not suitable for substitution or
replacement, including reasonable conservative or non-conservative
substitutions.
[0102] In this regard, and with exemplary reference to PlySs2 lysin
it is pointed out that, although the PlySs2 polypeptide lysin
represents a divergent class of prophage lytic enzyme, the lysin
comprises an N-terminal CHAP domain (cysteine-histidine
amidohydrolase/peptidase) (SEQ ID NO: 3) and a C-terminal SH3-type
5 domain (SEQ ID NO: 4) as depicted in FIG. 29. The domains are
depicted in the amino acid sequence in distinct shaded color
regions, with the CHAP domain corresponding to the first shaded
amino acid sequence region starting with LNN . . . and the SH3-type
5 domain corresponding to the second shaded region starting with
RSY . . . CHAP domains are included in several previously
characterized streptococcal and staphylococcal phage lysins. Thus,
one of skill in the art can reasonably make and test substitutions
or replacements to the CHAP domain and/or the SH-3 domain of
PlySs2. Sequence comparisons to the Genbank database can be made
with either or both of the CHAP and/or SH-3 domain sequences or
with the PlySs2 lysin full amino acid sequence, for instance, to
identify amino acids for substitution. For example, the CHAP domain
contains conserved cysteine and histidine amino acid sequences (the
first cysteine and histidine in the CHAP domain) which are
characteristic and conserved in CHAP domains of different
polypeptides. It is reasonable to predict, for example, that the
conserved cysteine and histidine residues should be maintained in a
mutant or variant of PlySs2 so as to maintain activity or
capability. It is notable that a mutant or variant having an
alanine replaced for valine at valine amino acid 19 in the PlySs2
amino acid sequence of FIG. 29 (SEQ ID NO: 1) is active and capable
of killing gram positive bacteria in a manner similar to and as
effective as the FIG. 29 (SEQ ID NO: 1) PlySs2 lysin.
[0103] The following is one example of various groupings of amino
acids:
Amino Acids with Nonpolar R Groups
Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,
Tryptophan, Methionine
[0104] Amino Acids with Uncharged Polar R Groups
Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine,
Glutamine
[0105] Amino Acids with Charged Polar R Groups (Negatively Charged
at pH 6.0) Aspartic acid, Glutamic acid
Basic Amino Acids (Positively Charged at pH 6.0)
Lysine, Arginine, Histidine (at pH 6.0)
[0106] Another grouping may be those amino acids with phenyl
groups: Phenylalanine, Tryptophan, Tyrosine
[0107] Another grouping may be according to molecular weight (i.e.,
size of R groups):
TABLE-US-00002 Glycine 75 Alanine 89 Serine 105 Proline 115 Valine
117 Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131
Asparagine 132 Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic
acid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165
Arginine 174 Tyrosine 181 Tryptophan 204
[0108] Particularly preferred substitutions are:
[0109] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0110] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0111] Ser for Thr such that a free--OH can be maintained; and
[0112] Gln for Asn such that a free NH.sub.2 can be maintained.
[0113] Exemplary and preferred conservative amino acid
substitutions include any of: glutamine (Q) for glutamic acid (E)
and vice versa; leucine (L) for valine (V) and vice versa; serine
(S) for threonine (T) and vice versa; isoleucine (I) for valine (V)
and vice versa; lysine (K) for glutamine (Q) and vice versa;
isoleucine (I) for methionine (M) and vice versa; serine (S) for
asparagine (N) and vice versa; leucine (L) for methionine (M) and
vice versa; lysine (L) for glutamic acid (E) and vice versa;
alanine (A) for serine (S) and vice versa; tyrosine (Y) for
phenylalanine (F) and vice versa; glutamic acid (E) for aspartic
acid (D) and vice versa; leucine (L) for isoleucine (I) and vice
versa; lysine (K) for arginine (R) and vice versa.
[0114] Amino acid substitutions may also be introduced to
substitute an amino acid with a particularly preferable property.
For example, a Cys may be introduced a potential site for disulfide
bridges with another Cys. A His may be introduced as a particularly
"catalytic" site (i.e., His can act as an acid or base and is the
most common amino acid in biochemical catalysis). Pro may be
introduced because of its particularly planar structure, which
induces .beta.-turns in the protein's structure.
[0115] In accordance with the present invention compositions and
methods are provide based on combinations of bacteriophage lysin(s)
with antibiotic are provided for rapid and effective killing of
gram positive bacteria. In accordance with the invention, the lysin
PlySs2, which demonstrates broad killing activity against multiple
bacteria, particularly gram-positive bacteria, including
Staphylococcus and Streptococcus bacterial strains, provides
remarkable synergy in combination with antibiotic(s) and can
significantly reduce the effective MIC doses required for
antibiotic(s).
[0116] As demonstrated and provided herein, lysin particularly
PlySs2 lysin is capable of synergizing with antibiotics, including
antibiotics of different types and classes, including vancomycin,
daptomycin, linezolid, and oxacillin, in a process characterized by
improved bactericidal activity, more rapid antibiotic penetration,
and suppression of resistance. In murine bacteremia models, as
demonstrated herein, pair-wise combinations of PlySs2 with
antibiotics confer a highly significant survival increase relative
to single-agent treatments. Thus, lysin/antibiotic combinations,
relative to current standard treatments, will be more effective
therapies for treating bacteremia in the clinic.
[0117] The invention further demonstrates PlySs2-dependent
enhancement of antibiotics in combination via both in-vitro assays
and in a murine model of S. aureus-induced bacteremia under
conditions in which human-simulated doses of single-agent
antibiotics fail. Data are presented herein illustrating the
mechanism of the PlySs2-mediated enhancement of antibiotic activity
and indicating a general synergy between lysins and antibiotics.
Synergy has implications for an efficacious new general
anti-infective strategy based on the co-administration of lysin and
antibiotics. In particular each and both agents lysins and
antibiotics may be administered at significantly reduced doses and
amounts, with enhanced bacteriocidal and bacteriostatic activity
and with reduced risk of antibiotic or agent resistance.
[0118] While lysin, particularly PlySs2 lysin, is recognized as a
single agent, the present invention provides that lysin,
particularly PlySs2 lysin, remarkably demonstrates a significant
degree of in vitro and in vivo synergy with various antibiotics.
While in the present Examples synergy is validated by time-kill
curves and checkerboard assays with multiple strains and
antibiotics, the extent of in vitro synergy is particularly
illustrated using a dual agent MIC assay in which as little as
0.25.times. MIC PlySs2 reduced the daptomycin MIC from 1 .mu.g/mL
to 0.0075 .mu.g/mL, a 128-fold decrease. This synergistic effect
was seen across 12 MRSA strains with the degree of potency
enhancement ranging from 64 to 256-fold. The two antimicrobials,
antibiotics plus lysin, in a combination are therefore doing more
than simply killing sequentially (reduction of the bulk population
by lysin followed by antibiotic killing of residual bacteria) since
7.5 ng/ml daptomycin is vastly insufficient to kill as a single
agent.
[0119] In the bacteremia models provided and demonstrated herein,
combination therapy treatments consistently outperformed full
strength human-simulated doses of single agent antibiotic
treatments. This is demonstrated for both vancomycin and
daptomycin, the current standard-of-care antibiotics for treating
MRSA bacteremia, as well as for oxacillin, a beta-lactam, the
current standard-of-care antibiotic for treating MSSA bacteremia.
These results have clear clinical implications and provide new
effective combination therapy regimens employing lysin(s) and
antibiotic(s) for treating bacteremia as well as other serious
infections. Provided are methods and compositions based on
combination lysin plus antibiotic therapy using lower doses of
these agents with enhanced efficacy and lower risk of resistance.
Indeed the present methods and compositions are effective on
resistant bacteria, including antibiotic resistant Staphylococcal
bacteria.
[0120] In clinical applications, the invention provides methods of
treating bacteremia by administering a lysin/antibiotic
combination, particularly PlySs2/antibiotic combination. While
above its MIC, the fast-acting lysin will effectively reduce the
pathogen population. Once the lysin concentration falls below the
MIC, the combination partner antibiotic's activity will be enhanced
synergistically by the presence of the lysin for approximately one
or two more lysin pharmacokinetic half-lives extending the time in
which synergy-enhanced killing is active. Thus, PlySs2/antibiotic
combinations will provide more potent and effective antibacterial
therapies than the currently available single-agent options.
[0121] The PlySs2 lysin displays activity and capability to kill
numerous distinct strains and species of gram positive bacteria,
including Staphylococcal, Streptococcal, Listeria, or Enterococcal
bacteria. In particular and with significance, PlySs2 is active in
killing Staphylococcus strains, including Staphylococcus aureus,
particularly both antibiotic-sensitive and distinct
antibiotic-resistant strains. PlySs2 is also active in killing
Streptococcus strains, and shows particularly effective killing
against Group A and Group B streptococcus strains. PlySs2 lysin
capability against bacteria is depicted below in TABLE 1, based on
log kill assessments using isolated strains in vitro.
TABLE-US-00003 TABLE 1 PlySs2 Reduction in Growth of Different
Bacteria (partial listing) Bacteria Relative Kill with PlySs2
Staphylococcus aureus +++ (VRSA, VISA, MRSA, MSSA) Streptococcus
suis +++ Staphylococcus epidermidis ++ Staphylococcus simulans +++
Lysteria monocytogenes ++ Enterococcus faecalis ++ Streptococcus
dysgalactiae - GBS ++ Streptococcus agalactiae -GBS +++
Streptococcus pyogenes -GAS +++ Streptococcus equi ++ Streptococcus
sanguinis ++ Streptococcus gordonii ++ Streptococcus sobrinus +
Streptococcus rattus + Streptococcus oralis + Streptococcus
pneumonine + Bacillus thuringiensis - Bacillus cereus - Bacillus
subtilis - Bacillus anthracis - Escherichia coli - Enterococcus
faecium - Pseudomanas aeruginosa -
[0122] 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.
A monoclonal antibody thus typically displays a single binding
affinity for any antigen with which it immunoreacts. A monoclonal
antibody may therefore contain an antibody molecule having a
plurality of antibody combining sites, each immunospecific for a
different antigen; e.g., a bispecific (chimeric) monoclonal
antibody.
[0123] The term "specific" may be used to refer to the situation in
which one member of a specific binding pair will not show
significant binding to molecules other than its specific binding
partner(s). The term is also applicable where e.g. an antigen
binding domain is specific for a particular epitope which is
carried by a number of antigens, in which case the specific binding
member carrying the antigen binding domain will be able to bind to
the various antigens carrying the epitope.
[0124] The term "comprise" generally used in the sense of include,
that is to say permitting the presence of one or more features or
components.
[0125] The term "consisting essentially of" refers to a product,
particularly a peptide sequence, of a defined number of residues
which is not covalently attached to a larger product. In the case
of the peptide of the invention hereof, those of skill in the art
will appreciate that minor modifications to the N- or C-terminal of
the peptide may however be contemplated, such as the chemical
modification of the terminal to add a protecting group or the like,
e.g. the amidation of the C-terminus.
[0126] The term "isolated" refers to the state in which the lysin
polypeptide(s) of the invention, or nucleic acid encoding such
polypeptides will be, in accordance with the present invention.
Polypeptides and nucleic acid will be free or substantially free of
material with which they are naturally associated such as other
polypeptides or nucleic acids with which they are found in their
natural environment, or the environment in which they are prepared
(e.g. cell culture) when such preparation is by recombinant DNA
technology practiced in vitro or in vivo. Polypeptides and nucleic
acid may be formulated with diluents or adjuvants and still for
practical purposes be isolated--for example the polypeptides will
normally be mixed with polymers or mucoadhesives or other carriers,
or will be mixed with pharmaceutically acceptable carriers or
diluents, when used in diagnosis or therapy.
[0127] Nucleic acids capable of encoding the S. suis PlySs2 lysin
polypeptide(s) useful and applicable in the invention are provided
herein. Representative nucleic acid sequences in this context are
polynucleotide sequences coding for the polypeptide of FIG. 29 (SEQ
ID NO: 1), and sequences that hybridize, under stringent
conditions, with complementary sequences of the DNA of the FIG. 29
(SEQ ID NO: 2) sequence(s). Further variants of these sequences and
sequences of nucleic acids that hybridize with those shown in the
figures also are contemplated for use in production of lysing
enzymes according to the disclosure, including natural variants
that may be obtained. A large variety of isolated nucleic acid
sequences or cDNA sequences that encode phage associated lysing
enzymes and partial sequences that hybridize with such gene
sequences are useful for recombinant production of the lysin
enzyme(s) or polypeptide(s) of the invention.
[0128] A "replicon" is any genetic element (e.g., plasmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo; i.e., capable of replication under its own
control.
[0129] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment.
[0130] A "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).
[0131] An "origin of replication" refers to those DNA sequences
that participate in DNA synthesis.
[0132] 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 coding 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,
genomic 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.
[0133] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0134] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA"
boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno
sequences in addition to the -10 and -35 consensus sequences.
[0135] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "under the control" of
transcriptional and translational control sequences in a cell when
RNA polymerase transcribes the coding sequence into mRNA, which is
then translated into the protein encoded by the coding
sequence.
[0136] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0137] The term "oligonucleotide," as used herein in referring to
the probe of the present invention, is defined as a molecule
comprised of two or more ribonucleotides, preferably more than
three. Its exact size will depend upon many factors which, in turn,
depend upon the ultimate function and use of the
oligonucleotide.
[0138] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0139] A cell has been "transformed" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into chromosomal DNA making up the genome of the cell. In
prokaryotes, yeast, and mammalian cells for example, the
transforming DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
comprised of a population of daughter cells containing the
transforming DNA. A "clone" is a population of cells derived from a
single cell or common ancestor by mitosis. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for
many generations.
[0140] Two DNA sequences are "substantially homologous" when at
least about 75% (preferably at least about 80%, and most preferably
at least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I &
II, supra; Nucleic Acid Hybridization, supra.
[0141] DNA molecules and nucleotide sequences which are derivatives
of those specifically disclosed herein and which differ from those
disclosed by the deletion, addition or substitution of nucleotides
while still encoding a protein which possesses the functional
characteristic of the lysin polypeptide(s) are contemplated by the
disclosure. Also included are small DNA molecules which are derived
from the disclosed DNA molecules. Such small DNA molecules include
oligonucleotides suitable for use as hybridization probes or
polymerase chain reaction (PCR) primers. As such, these small DNA
molecules will comprise at least a segment of a lytic enzyme
genetically coded for by a bacteriophage of Staphylococcus suis
and, for the purposes of PCR, will comprise at least a 10-15
nucleotide sequence and, more preferably, a 15-30 nucleotide
sequence of the gene. DNA molecules and nucleotide sequences which
are derived from the disclosed DNA molecules as described above may
also be defined as DNA sequences which hybridize under stringent
conditions to the DNA sequences disclosed, or fragments
thereof.
[0142] In preferred embodiments of the present disclosure,
stringent conditions may be defined as those under which DNA
molecules with more than 25% sequence variation (also termed
"mismatch") will not hybridize. In a more preferred embodiment,
stringent conditions are those under which DNA molecules with more
than 15% mismatch will not hybridize, and more preferably still,
stringent conditions are those under which DNA sequences with more
than 10% mismatch will not hybridize. Preferably, stringent
conditions are those under which DNA sequences with more than 6%
mismatch will not hybridize.
[0143] The degeneracy of the genetic code further widens the scope
of the embodiments as it enables major variations in the nucleotide
sequence of a DNA molecule while maintaining the amino acid
sequence of the encoded protein. Thus, the nucleotide sequence of
the gene could be changed at this position to any of these three
codons without affecting the amino acid composition of the encoded
protein or the characteristics of the protein. The genetic code and
variations in nucleotide codons for particular amino acids are well
known to the skilled artisan. Based upon the degeneracy of the
genetic code, variant DNA molecules may be derived from the cDNA
molecules disclosed herein using standard DNA mutagenesis
techniques as described above, or by synthesis of DNA sequences.
DNA sequences which do not hybridize under stringent conditions to
the cDNA sequences disclosed by virtue of sequence variation based
on the degeneracy of the genetic code are herein comprehended by
this disclosure.
[0144] Thus, it should be appreciated that also within the scope of
the present invention are DNA sequences encoding a lysin of the
present invention, including PlySs2 and PlySs1, which sequences
code for a polypeptide having the same amino acid sequence as
provided in FIG. 29 (SEQ ID NO: 1), but which are degenerate
thereto or are degenerate to the exemplary nucleic acids sequences
provided in FIG. 29 (SEQ ID NO: 2). By "degenerate to" is meant
that a different three-letter codon is used to specify a particular
amino acid. It is well known in the art the codons which can be
used interchangeably to code for each specific amino acid.
[0145] One skilled in the art will recognize that the DNA
mutagenesis techniques described here and known in the art can
produce a wide variety of DNA molecules that code for a
bacteriophage lysin of Streptococcus suis yet that maintain the
essential characteristics of the lytic polypeptides described and
provided herein. Newly derived proteins may also be selected in
order to obtain variations on the characteristic of the lytic
polypeptide(s), as will be more fully described below. Such
derivatives include those with variations in amino acid sequence
including minor deletions, additions and substitutions.
[0146] While the site for introducing an amino acid sequence
variation may be predetermined, the mutation per se does not need
to be predetermined Amino acid substitutions are typically of
single residues, or can be of one or more, one or a few, one, two,
three, four, five, six or seven residues; insertions usually will
be on the order of about from 1 to 10 amino acid residues; and
deletions will range about from 1 to 30 residues. Deletions or
insertions may be in single form, but preferably are made in
adjacent pairs, i.e., a deletion of 2 residues or insertion of 2
residues. Substitutions, deletions, insertions or any combination
thereof may be combined to arrive at a final construct.
Substitutional variants are those in which at least one residue in
the amino acid sequence has been removed and a different residue
inserted in its place. Such substitutions may be made so as to
generate no significant effect on the protein characteristics or
when it is desired to finely modulate the characteristics of the
protein Amino acids which may be substituted for an original amino
acid in a protein and which are regarded as conservative
substitutions are described above and will be recognized by one of
skill in the art.
[0147] As is well known in the art, DNA sequences may be expressed
by operatively linking them to an expression control sequence in an
appropriate expression vector and employing that expression vector
to transform an appropriate unicellular host. Such operative
linking of a DNA sequence of this invention to an expression
control sequence, of course, includes, if not already part of the
DNA sequence, the provision of an initiation codon, ATG, in the
correct reading frame upstream of the DNA sequence. A wide variety
of host/expression vector combinations may be employed in
expressing the DNA sequences of this invention. Useful expression
vectors, for example, may consist of segments of chromosomal,
non-chromosomal and synthetic DNA sequences. Any of a wide variety
of expression control sequences--sequences that control the
expression of a DNA sequence operatively linked to it--may be used
in these vectors to express the DNA sequences of this invention. A
wide variety of unicellular host cells are also useful in
expressing the DNA sequences of this invention. These hosts may
include well known eukaryotic and prokaryotic hosts, such as
strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such
as yeasts, and animal cells, human cells and plant cells in tissue
culture. One skilled in the art will be able to select the proper
vectors, expression control sequences, and hosts without undue
experimentation to accomplish the desired expression without
departing from the scope of this invention.
[0148] Therapeutic or pharmaceutical compositions comprising the
lytic enzyme(s)/polypeptide(s) of use in the methods and
applications provided in the invention are provided herein, as well
as related methods of use. Therapeutic or pharmaceutical
compositions may comprise one or more lytic polypeptide(s), and
optionally include natural, truncated, chimeric or shuffled lytic
enzymes, combined with one or more antibiotics, optionally combined
with suitable excipients, carriers or vehicles. The invention
provides therapeutic compositions or pharmaceutical compositions of
the lysins, including PlySs2, in combination with antibiotic for
use in the killing, alleviation, decolonization, prophylaxis or
treatment of gram-positive bacteria, including bacterial infections
or related conditions. The invention provides therapeutic
compositions or pharmaceutical compositions of the lysins,
including PlySs2, in combination with vancomycin, linezolid or
daptomycin for use in the killing, alleviation, decolonization,
prophylaxis or treatment of gram-positive bacteria, including
bacterial infections or related conditions. The invention provides
therapeutic compositions or pharmaceutical compositions of the
lysins, including PlySs2, in combination with daptomycin for use in
the killing, alleviation, decolonization, prophylaxis or treatment
of gram-positive bacteria, including bacterial infections or
related conditions. Compositions comprising PlySs2 lysin, including
truncations or variants thereof, in combination with antibiotic,
including daptomycin, are provided herein for use in the killing,
alleviation, decolonization, prophylaxis or treatment of
gram-positive bacteria, including bacterial infections or related
conditions, particularly of Streptococcus, Staphylococcus,
Enterococcus or Listeria, including Streptococcus pyogenes and
antibiotic resistant Staphylococcus aureus.
[0149] The enzyme(s) or polypeptide(s) included in the therapeutic
compositions may be one or more or any combination of unaltered
phage associated lytic enzyme(s), truncated lytic polypeptides,
variant lytic polypeptide(s), and chimeric and/or shuffled lytic
enzymes. Additionally, different lytic polypeptide(s) genetically
coded for by different phage for treatment of the same bacteria may
be used. These lytic enzymes may also be any combination of
"unaltered" lytic enzymes or polypeptides, truncated lytic
polypeptide(s), variant lytic polypeptide(s), and chimeric and
shuffled lytic enzymes. The lytic enzyme(s)/polypeptide(s) in a
therapeutic or pharmaceutical composition for gram-positive
bacteria, including Streptococcus, Staphylococcus, Enterococcus and
Listeria, may be used alone or in combination with antibiotics or,
if there are other invasive bacterial organisms to be treated, in
combination with other phage associated lytic enzymes specific for
other bacteria being targeted. The lytic enzyme, truncated enzyme,
variant enzyme, chimeric enzyme, and/or shuffled lytic enzyme may
be used in conjunction with a holin protein. The amount of the
holin protein may also be varied. Various antibiotics may be
optionally included in the therapeutic composition with the
enzyme(s) or polypeptide(s) and with or without the presence of
lysostaphin. More than one lytic enzyme or polypeptide may be
included in the therapeutic composition.
[0150] The pharmaceutical composition can also include one or more
altered lytic enzymes, including isozymes, analogs, or variants
thereof, produced by chemical synthesis or DNA recombinant
techniques. In particular, altered lytic protein can be produced by
amino acid substitution, deletion, truncation, chimerization,
shuffling, or combinations thereof. The pharmaceutical composition
may contain a combination of one or more natural lytic protein and
one or more truncated, variant, chimeric or shuffled lytic protein.
The pharmaceutical composition may also contain a peptide or a
peptide fragment of at least one lytic protein derived from the
same or different bacteria species, with an optional addition of
one or more complementary agent, and a pharmaceutically acceptable
carrier or diluent.
[0151] The pharmaceutical compositions of the present invention
contain a complementary agent--one or more conventional
antibiotics--particularly as provided herein. Antibiotics can be
subgrouped broadly into those affecting cell wall peptidoglycan
biosynthesis and those affecting DNA or protein synthesis in gram
positive bacteria. Cell wall synthesis inhibitors, including
penicillin and antibiotics like it, disrupt the rigid outer cell
wall so that the relatively unsupported cell swells and eventually
ruptures. The complementary agent may be an antibiotic, such as
erythromycin, clarithromycin, azithromycin, roxithromycin, other
members of the macrolide family, penicilins, cephalosporins, and
any combinations thereof in amounts which are effective to
synergistically enhance the therapeutic effect of the lytic enzyme.
Virtually any other antibiotic may be used with the altered and/or
unaltered lytic enzyme. Antibiotics affecting cell wall
peptidoglycan biosynthesis include: Glycopeptides, which inhibit
peptidoglycan synthesis by preventing the incorporation of
N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide
subunits into the peptidoglycan matrix. Available glycopeptides
include vancomycin and teicoplanin.; Penicillins, which act by
inhibiting the formation of peptidoglycan cross-links. The
functional group of penicillins, the .beta.-lactam moiety, binds
and inhibits DD-transpeptidase that links the peptidoglycan
molecules in bacteria. Hydrolytic enzymes continue to break down
the cell wall, causing cytolysis or death due to osmotic pressure.
Common penicillins include oxacillin, ampicillin and cloxacillin;
and Polypeptides, which interfere with the dephosphorylation of the
C.sub.55-isoprenyl pyrophosphate, a molecule that carries
peptidoglycan building-blocks outside of the plasma membrane. A
cell wall-impacting polypeptide is bacitracin. Other useful and
relevant antibiotics include vancomycin, linezolid, and
daptomycin.
[0152] Other lytic enzymes may be included in the carrier to treat
other bacterial infections. The pharmaceutical composition can also
contain a peptide or a peptide fragment of at least one lytic
protein, one holin protein, or at least one holin and one lytic
protein, which lytic and holin proteins are each derived from the
same or different bacteria species, with an optional addition of a
complementary agents, and a suitable carrier or diluent.
[0153] Also provided are compositions containing nucleic acid
molecules that, either alone or in combination with other nucleic
acid molecules, are capable of expressing an effective amount of a
lytic polypeptide(s) or a peptide fragment of a lytic
polypeptide(s) in vivo. Cell cultures containing these nucleic acid
molecules, polynucleotides, and vectors carrying and expressing
these molecules in vitro or in vivo, are also provided.
[0154] Therapeutic or pharmaceutical compositions may comprise
lytic polypeptide(s) and antibiotic(s) combined with a variety of
carriers to treat the illnesses caused by the susceptible
gram-positive bacteria. The carrier suitably contains minor amounts
of additives such as substances that enhance isotonicity and
chemical stability. Such materials are non-toxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, succinate, acetic acid, and other organic
acids or their salts; antioxidants such as ascorbic acid; low
molecular weight (less than about ten residues) polypeptides, e.g.,
polyarginine or tripeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; glycine; amino acids such as glutamic acid,
aspartic acid, histidine, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, trehalose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as mannitol or sorbitol;
counter-ions such as sodium; non-ionic surfactants such as
polysorbates, poloxamers, or polyethylene glycol (PEG); and/or
neutral salts. Glycerin or glycerol (1,2,3-propanetriol) is
commercially available for pharmaceutical use. DMSO is an aprotic
solvent with a remarkable ability to enhance penetration of many
locally applied drugs. The carrier vehicle may also include
Ringer's solution, a buffered solution, and dextrose solution,
particularly when an intravenous solution is prepared.
[0155] The effective dosage rates or amounts of an altered or
unaltered lytic enzyme/polypeptide(s) of and for use in the present
invention will depend in part on whether the lytic
enzyme/polypeptide(s) will be used therapeutically or
prophylactically, the duration of exposure of the recipient to the
infectious bacteria, the size and weight of the individual, etc.
The duration for use of the composition containing the
enzyme/polypeptide(s) also depends on whether the use is for
prophylactic purposes, wherein the use may be hourly, daily or
weekly, for a short time period, or whether the use will be for
therapeutic purposes wherein a more intensive regimen of the use of
the composition may be needed, such that usage may last for hours,
days or weeks, and/or on a daily basis, or at timed intervals
during the day. Any dosage form employed should provide for a
minimum number of units for a minimum amount of time. Carriers that
are classified as "long" or "slow" release carriers (such as, for
example, certain nasal sprays or lozenges) could possess or provide
a lower concentration of active (enzyme) units per ml, but over a
longer period of time, whereas a "short" or "fast" release carrier
(such as, for example, a gargle) could possess or provide a high
concentration of active (enzyme) units per ml, but over a shorter
period of time. The amount of active units per ml and the duration
of time of exposure depend on the nature of infection, whether
treatment is to be prophylactic or therapeutic, and other
variables. There are situations where it may be necessary to have a
much higher unit/ml dosage or a lower unit/ml dosage.
[0156] The lytic enzyme/polypeptide(s) should be in an environment
having a pH which allows for activity of the lytic
enzyme/polypeptide(s). A stabilizing buffer may allow for the
optimum activity of the lysin enzyme/polypeptide(s). The buffer may
contain a reducing reagent, such as dithiothreitol or beta
mercaptoethanol (BME). The stabilizing buffer may also be or
include a metal chelating reagent, such as
ethylenediaminetetracetic acid disodium salt, or it may also
contain a phosphate or citrate-phosphate buffer, or any other
buffer.
[0157] It is notable that the environment and certain aspects of
the treatment location can affect the effectiveness of an
antibiotic. For instance, daptomycin binds avidly to pulmonary
surfactant and therefore is not effective in treatment of bacterial
pneumonia, including staphylococcal pneumonia. The present
invention demonstrates the remarkable effectiveness and synergy of
PlySs2 and daptomycin in combination against susceptible bacteria.
In addition, PlySs2 lysin and daptomycin in combination remain very
effective in the presence of a commercially available surfactant
which mimics pulmonary surfactant. Thus, PlySs2 facilitates and
enhances the effectiveness of antibiotic, particularly daptomycin,
and serves to enable daptomycin effectiveness and activity even in
the presence of surfactant.
[0158] A mild surfactant can be included in a therapeutic or
pharmaceutical composition in an amount effective to potentiate the
therapeutic effect of the lytic enzyme/polypeptide(s) may be used
in a composition. Suitable mild surfactants include, inter alia,
esters of polyoxyethylene sorbitan and fatty acids (Tween series),
octylphenoxy polyethoxy ethanol (Triton-X series),
n-Octyl-.beta.-D-glucopyranoside,
n-Octyl-.beta.-D-thioglucopyranoside,
n-Decyl-.beta.-D-glucopyranoside,
n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurring
surfactants, e.g., fatty acids, glycerides, monoglycerides,
deoxycholate and esters of deoxycholate.
[0159] Preservatives may also be used in this invention and
preferably comprise about 0.05% to 0.5% by weight of the total
composition. The use of preservatives assures that if the product
is microbially contaminated, the formulation will prevent or
diminish microorganism growth. Some preservatives useful in this
invention include methylparaben, propylparaben, butylparaben,
chloroxylenol, sodium benzoate, DMDM Hydantoin,
3-Iodo-2-Propylbutyl carbamate, potassium sorbate, chlorhexidine
digluconate, or a combination thereof.
[0160] The therapeutic composition may further comprise other
enzymes, such as the enzyme lysostaphin for the treatment of any
Staphylococcus aureus bacteria present along with the susceptible
gram-positive bacteria. Lysostaphin, a gene product of
Staphylococcus simulans, exerts a bacteriostatic and bactericidal
effect upon S. aureus by enzymatically degrading the polyglycine
crosslinks of the cell wall (Browder et al., Res. Comm, 19: 393-400
(1965)). The gene for lysostaphin has subsequently been cloned and
sequenced (Recsei et al., Proc. Natl. Acad. Sci. USA, 84: 1127-1131
(1987). A therapeutic composition may also include mutanolysin, and
lysozyme.
[0161] Means of application of the therapeutic composition
comprising a lytic enzyme/polypeptide(s) and antibiotic(s) include,
but are not limited to direct, indirect, carrier and special means
or any combination of means. Direct application of the lytic
enzyme/polypeptide(s) may be by any suitable means to directly
bring the polypeptide in contact with the site of infection or
bacterial colonization, such as to the nasal area (for example
nasal sprays), dermal or skin applications (for example topical
ointments or formulations), suppositories, tampon applications,
etc. Nasal applications include for instance nasal sprays, nasal
drops, nasal ointments, nasal washes, nasal injections, nasal
packings, bronchial sprays and inhalers, or indirectly through use
of throat lozenges, mouthwashes or gargles, or through the use of
ointments applied to the nasal nares, or the face or any
combination of these and similar methods of application. The forms
in which the lytic enzyme may be administered include but are not
limited to lozenges, troches, candies, injectants, chewing gums,
tablets, powders, sprays, liquids, ointments, and aerosols.
[0162] The mode of application for the lytic enzyme and antibiotic
includes a number of different types and combinations of carriers
which include, but are not limited to an aqueous liquid, an alcohol
base liquid, a water soluble gel, a lotion, an ointment, a
nonaqueous liquid base, a mineral oil base, a blend of mineral oil
and petrolatum, lanolin, liposomes, protein carriers such as serum
albumin or gelatin, powdered cellulose carmel, and combinations
thereof. A mode of delivery of the carrier containing the
therapeutic agent includes, but is not limited to a smear, spray, a
time-release patch, a liquid absorbed wipe, and combinations
thereof. The lytic enzyme may be applied to a bandage either
directly or in one of the other carriers. The bandages may be sold
damp or dry, wherein the enzyme is in a lyophilized form on the
bandage. This method of application is most effective for the
treatment of infected skin. The carriers of topical compositions
may comprise semi-solid and gel-like vehicles that include a
polymer thickener, water, preservatives, active surfactants or
emulsifiers, antioxidants, sun screens, and a solvent or mixed
solvent system Polymer thickeners that may be used include those
known to one skilled in the art, such as hydrophilic and
hydroalcoholic gelling agents frequently used in the cosmetic and
pharmaceutical industries. Other preferred gelling polymers include
hydroxyethylcellulose, cellulose gum, MVE/MA decadiene
crosspolymer, PVM/MA copolymer, or a combination thereof.
[0163] A composition comprising a lytic enzyme/polypeptide(s) and
antibiotic(s) can be administered in the form of a candy, chewing
gum, lozenge, troche, tablet, a powder, an aerosol, a liquid, a
liquid spray, or toothpaste for the prevention or treatment of
bacterial infections associated with upper respiratory tract
illnesses. The lozenge, tablet, or gum into which the lytic
enzyme/polypeptide(s) is added may contain sugar, corn syrup, a
variety of dyes, non-sugar sweeteners, flavorings, any binders, or
combinations thereof. Similarly, any gum-based products may contain
acacia, carnauba wax, citric acid, cornstarch, food colorings,
flavorings, non-sugar sweeteners, gelatin, glucose, glycerin, gum
base, shellac, sodium saccharin, sugar, water, white wax,
cellulose, other binders, and combinations thereof. Lozenges may
further contain sucrose, cornstarch, acacia, gum tragacanth,
anethole, linseed, oleoresin, mineral oil, and cellulose, other
binders, and combinations thereof. Sugar substitutes can also be
used in place of dextrose, sucrose, or other sugars. Compositions
comprising lytic enzymes, or their peptide fragments can be
directed to the mucosal lining, where, in residence, they kill
colonizing disease bacteria. The mucosal lining, as disclosed and
described herein, includes, for example, the upper and lower
respiratory tract, eye, buccal cavity, nose, rectum, vagina,
periodontal pocket, intestines and colon. Due to natural
eliminating or cleansing mechanisms of mucosal tissues,
conventional dosage forms are not retained at the application site
for any significant length of time.
[0164] It may be advantageous to have materials which exhibit
adhesion to mucosal tissues, to be administered with one or more
phage enzymes and other complementary agents over a period of time.
Materials having controlled release capability are particularly
desirable, and the use of sustained release mucoadhesives has
received a significant degree of attention. Other approaches
involving mucoadhesives which are the combination of hydrophilic
and hydrophobic materials, are known. Micelles and multilamillar
micelles may also be used to control the release of enzyme.
Materials having capacity to target or adhere to surfaces, such as
plastic, membranes, devices utilized in clinical practice,
including particularly any material or component which is placed in
the body and susceptible to bacterial adhesion or biofilm
development, such as catheters, valves, prosthetic devices, drug or
compound pumps, stents, orthopedic materials, etc, may be combined,
mixed, or fused to the lysin(s) of use in the present
invention.
[0165] Therapeutic or pharmaceutical compositions of the invention
can also contain polymeric mucoadhesives including a graft
copolymer comprising a hydrophilic main chain and hydrophobic graft
chains for controlled release of biologically active agents. The
compositions of this application may optionally contain other
polymeric materials, such as poly(acrylic acid), poly,-(vinyl
pyrrolidone), and sodium carboxymethyl cellulose plasticizers, and
other pharmaceutically acceptable excipients in amounts that do not
cause deleterious effect upon mucoadhesivity of the
composition.
[0166] A lytic enzyme/polypeptide(s) and antibiotic(s) of the
invention may be administered for use in accordance with the
invention by any pharmaceutically applicable or acceptable means
including topically, orally or parenterally. For example, the lytic
enzyme/polypeptide(s) can be administered intramuscularly,
intrathecally, subdermally, subcutaneously, or intravenously to
treat infections by gram-positive bacteria. In cases where
parenteral injection is the chosen mode of administration, an
isotonic formulation is preferably used. Generally, additives for
isotonicity can include sodium chloride, dextrose, mannitol,
sorbitol and lactose. In some cases, isotonic solutions such as
phosphate buffered saline are preferred. Stabilizers include
gelatin and albumin A vasoconstriction agent can be added to the
formulation. The pharmaceutical preparations according to this
application are provided sterile and pyrogen free.
[0167] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model is
also used to achieve a desirable concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. The exact
dosage is chosen by the individual physician in view of the patient
to be treated. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired
effect. Additional factors which may be taken into account include
the severity of the disease state, age, weight and gender of the
patient; diet, desired duration of treatment, method of
administration, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long acting pharmaceutical compositions might be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0168] The effective dosage rates or amounts of the lytic
enzyme/polypeptide(s) to be administered, and the duration of
treatment will depend in part on the seriousness of the infection,
the weight of the patient, particularly human, the duration of
exposure of the recipient to the infectious bacteria, the number of
square centimeters of skin or tissue which are infected, the depth
of the infection, the seriousness of the infection, and a variety
of a number of other variables. The composition may be applied
anywhere from once to several times a day or a week, and may be
applied for a short, such as days or up to several weeks, or long
term period, such as many weeks or up to months. The usage may last
for days or weeks. Any dosage form employed should provide for a
minimum number of units for a minimum amount of time. The
concentration of the active units of enzymes believed to provide
for an effective amount or dosage of enzymes may be selected as
appropriate.
[0169] The lysin and antibiotics of use and application in the
compositions and methods of the invention may be administered
simultaneously or subsequently. The lysin and antibiotic agents may
be administered in a single dose or multiple doses, singly or in
combination. The lysin and antibiotic may be administered by the
same mode of administration or by different modes of
administration, and may be administered once, twice or multiple
times, one or more in combination or individually. Thus, lysin may
be administered in an initial dose followed by a subsequent dose or
doses, particularly depending on the response and bacterial killing
or decolonization, and may be combined or alternated with
antibiotic dose(s). In a particular aspect of the invention and in
view of the reduction in the development of resistance to
antibiotics by administering a lysin, particularly PlySs2, with
antibiotic, combinations of antibiotic and lysin may be
administered for longer periods and dosing can be extended without
risk of resistance. In addition, in as much as the doses required
for efficacy of each of antibiotic and lysin are significantly
reduced by combining or co-administering the agents simultaneously
or in series, a patient can be treated more aggressively and more
continually without risk, or with reduced risk, of resistance.
[0170] The term `agent` means any molecule, including polypeptides,
antibodies, polynucleotides, chemical compounds and small
molecules. In particular the term agent includes compounds such as
test compounds, added additional compound(s), or lysin enzyme
compounds.
[0171] The term `agonist` refers to a ligand that stimulates the
receptor the ligand binds to in the broadest sense.
[0172] The term `assay` means any process used to measure a
specific property of a compound. A `screening assay` means a
process used to characterize or select compounds based upon their
activity from a collection of compounds.
[0173] The term `preventing` or `prevention` refers to a reduction
in risk of acquiring or developing a disease or disorder (i.e.,
causing at least one of the clinical symptoms of the disease not to
develop) in a subject that may be exposed to a disease-causing
agent, or predisposed to the disease in advance of disease
onset.
[0174] The term `prophylaxis` is related to and encompassed in the
term `prevention`, and refers to a measure or procedure the purpose
of which is to prevent, rather than to treat or cure a disease.
Non-limiting examples of prophylactic measures may include the
administration of vaccines; the administration of low molecular
weight heparin to hospital patients at risk for thrombosis due, for
example, to immobilization; and the administration of an
anti-malarial agent such as chloroquine, in advance of a visit to a
geographical region where malaria is endemic or the risk of
contracting malaria is high.
[0175] `Therapeutically effective amount` means that amount of a
drug, compound, antimicrobial, antibody, polypeptide, or
pharmaceutical agent that will elicit the biological or medical
response of a subject that is being sought by a medical doctor or
other clinician. In particular, with regard to gram-positive
bacterial infections and growth of gram-positive bacteria, the term
"effective amount" is intended to include an effective amount of a
compound or agent that will bring about a biologically meaningful
decrease in the amount of or extent of infection of gram-positive
bacteria, including having a bacteriocidal and/or bacteriostatic
effect. The phrase "therapeutically effective amount" is used
herein to mean an amount sufficient to prevent, and preferably
reduce by at least about 30 percent, more preferably by at least 50
percent, most preferably by at least 90 percent, a clinically
significant change in the growth or amount of infectious bacteria,
or other feature of pathology such as for example, elevated fever
or white cell count as may attend its presence and activity.
[0176] The term `treating` or `treatment` of any disease or
infection refers, in one embodiment, to ameliorating the disease or
infection (i.e., arresting the disease or growth of the infectious
agent or bacteria or reducing the manifestation, extent or severity
of at least one of the clinical symptoms thereof). In another
embodiment `treating` or `treatment` refers to ameliorating at
least one physical parameter, which may not be discernible by the
subject. In yet another embodiment, `treating` or `treatment`
refers to modulating the disease or infection, either physically,
(e.g., stabilization of a discernible symptom), physiologically,
(e.g., stabilization of a physical parameter), or both. In a
further embodiment, `treating` or `treatment` relates to slowing
the progression of a disease or reducing an infection.
[0177] It is noted that in the context of treatment methods which
are carried out in vivo or medical and clinical treatment methods
in accordance with the present application and claims, the term
subject, patient or individual is intended to refer to a human.
[0178] The terms "gram-positive bacteria", "Gram-positive
bacteria", "gram-positive" and any variants not specifically
listed, may be used herein interchangeably, and as used throughout
the present application and claims refer to Gram-positive bacteria
which are known and/or can be identified by the presence of certain
cell wall and/or cell membrane characteristics and/or by staining
with Gram stain. Gram positive bacteria are known and can readily
be identified and may be selected from but are not limited to the
genera Listeria, Staphylococcus, Streptococcus, Enterococcus,
Mycobacterium, Corynebacterium, and Clostridium, and include any
and all recognized or unrecognized species or strains thereof. In
an aspect of the invention, the PlyS lysin sensitive gram-positive
bacteria include bacteria selected from one or more of Listeria,
Staphylococcus, Streptococcus, and Enterococcus.
[0179] The term "bacteriocidal" refers to capable of killing
bacterial cells.
[0180] The term "bacteriostatic" refers to capable of inhibiting
bacterial growth, including inhibiting growing bacterial cells.
[0181] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human.
[0182] The invention may be better understood by reference to the
following non-limiting Examples, which are provided as exemplary of
the invention. The following examples are presented in order to
more fully illustrate the preferred embodiments of the invention
and should in no way be construed, however, as limiting the broad
scope of the invention.
Example 1
[0183] PlySs2 lysin demonstrates the ability to kill various
strains of clinically significant gram-positive bacteria, including
antibiotic resistant strains such as methicillin and vancomycin
resistant and sensitive strains of Staphylococcus aureus (MRSA,
MSSA, VRSA, VISA), daptomycin-resistant Staphylococcus aureus
(DRSA), and linezolid-resistant Staphylococcus aureus (LRSA).
PlySs2 is a unique lysin in having broad species killing activity
and can kill multiple species of bacteria, particularly
gram-positive bacteria, including Staphylococcus, Streptococcus,
Enterococcus and Listeria bacterial strains. A tabulation of
sensitivity (as depicted using MIC values and uM concentrations) of
staphylococci to PlySs2 lysin and various antibiotics is shown in
TABLE 2. Minimally inhibitory concentration (MICs) were determined
using the broth microdilution method in accordance with standards
and as described in the Clinical and Laboratory Standards Institute
(CLSI) document M07-A9 (Methods for dilutional antimicrobial
sensitivity tests for bacteria that grow aerobically. Volume 32
(Wayne [PA]: Clinical and Laboratory Standards Institute [US],
2012). This value is the MIC determined in the presence of reducing
agent (such as DTT or BMS) in the MIC assay. Reducing agent is
added for the purpose of improving reproducibility between and
among assays in determining MIC values.
TABLE-US-00004 TABLE 2 PlySs2 and antibiotic activity against S.
aureus strains Organisms PlySs2 Daptomycin Vancomycin Oxacillin
Linezolid (#of strains) MIC.sub.90 [uM] MIC.sub.90 [uM] MIC.sub.90
[uM] MIC.sub.50/90 [uM] MIC.sub.50/90 [uM] MRSA 4 0.15 1 0.6 1 0.7
>4* >10.0 2 5.7 (n = 45) MSSA 4 0.15 1 0.6 1 0.7 n/a n/a 2
5.7 (n-28) VISA 32 1.2 8 4.9 4 2.7 nla n/a 2 5.7 (n = 10) VRSA 2
0.08 1 0.6 >16 >10.6 n/a n/a 2 5.7 (n = 14) LRSA 2 0.08 1 0.6
1 0.7 n/a n/a >64 >183 (n = 5) DRSA 4 0.15 16 9.9 1 0.7 n/a
n/a 2 5.7 (n = 8) *MICs were determined using the broth
microdilution method and evaluating 80% growth inhibition according
to CLSI methods (M07-A9). *Bold = drug failure (MIC value is above
EUCAST breakpoint for the indicated drug on S. aureus)
[0184] Activity of PlySs2 against various gram-positive and
gram-negative organisms is tabulated in TABLE 3, which includes MIC
values and range for the different organisms. Activity of PlySs2
against antibiotic-resistant Staphylococcus aureus is provided in
TABLE 4. PlySs2 has potent growth inhibitory activity on all
Staphylococcus aureus strains tested including 103 MSSA and 120
MRSA isolates (MIC=8 .mu.g/mL) as well as Groups A and B
streptococci and Staphylococcus lugdiensis (TABLE 3). Little or no
activity was observed on a collection of other Gram positive
bacteria as well as all Gram negative bacteria tested. Although
PlySs2 effectively kills antibiotic resistant and sensitive S.
aureus as well as numerous other clinically significant
gram-positive bacteria, it is notably ineffective on numerous
commensal bacteria, such as Escherichia coli, as shown above in
TABLE 1 and in TABLE 5 below which provides sensitivity of human
gut bacteria and PlySs2 MIC.
TABLE-US-00005 TABLE 3 Activity of PlySs2 Against Gram-Positive and
Gram-Negative Organisms Organism and susceptibility subset MIC
(.mu.g/mL) (no. tested) 50% 90% Range Staphylcoccus aureus
Methicillin susceptible (103) 4 8 1-16 Methicillin resistant (120)
4 8 1-16 Streptococcus pyogenes, Group A (54) 2 8 0.5-8
Streptococcus agalactiae, Group B (51) 8 16 1-64 Other
Gram-positive organisms Staphylococcus lugdiensis (10) 8 8 8
Staphylococcus epidermidis (11) 128 512 4-512 Streptococcus
pneumoniae (26) 16 64 1-64 Streptococcus mutans (12) 64 256 2-256
Listeria monocytogenes (12) 128 512 1-512 Enterococcus faecalis
(17) >512 >512 32->512 Enterococcus faecium (5) >512
>512 32->512 Bacillus cereus (10) >512 >512 >512
Gram-negative organisms Acinetobacter baumannii (8) >512 >512
>512 Escherichia coli (6) >512 >512 >512 Pseudomonas
aeruginosa (5) >512 >512 >512
TABLE-US-00006 TABLE 4 Activity of PlySs2 Against Antibiotic-
Resistant Staphylococcus aureus MIC (mg/mL) Susceptibility subset
(no. tested) 50% 90% Range Vancomycin-resistant (14) 2 4 1-4
Vancomycin-intermediate (31) 8 32 1-64 Linezolid-resistant (5) 2 2
2-4 Daptomycin-resistant (8) 2 4 2-4
TABLE-US-00007 TABLE 5 Sensitivity of Human Gut Bacteria to PlySs2
Organism N CF-301 MIC (ug/ml) Salmonella enteriditis 1 >512
Pseudomonas aeruginosa 11 >512 Escherichia coli 10 >512
Klebsiella spp. 8 >512 Proteus mirabilis 2 >512 Lactobacillus
spp. 6 >512 Lactococcus spp. 3 >512
[0185] While PlySs2 is effective against many different clinically
relevant bacteria, it retains the beneficial character of many
lysins in lacking broad spectrum bacterial killing, therefore side
effects such as intestinal flora disturbance seen with many
antibiotics will be minimized. In addition, lysins have
demonstrated a low probability of bacterial resistance. PlySs2's
remarkably broad clinically relevant killing capability make it
uniquely applicable to the clinical setting, including in instances
where there is a fully uncharacterized or mixed bacterial
infection.
[0186] Staphylococcus aureus is the causative agent of several
serious infectious diseases and the emergence of antibiotic
resistant S. aureus strains has resulted in significant treatment
difficulties, intensifying the need for new antimicrobial agents.
Currently, 40 to 60% of nosocomial infections of S. aureus are
resistant to oxacillin (Massey R C et al (2006) Nat Rev Microbiol
4:953-958), and greater than 60% of the isolates are resistant to
methicillin (Gill S R et al (2005) J Bacteriol 187:2426-2438). A
number of new antimicrobial agents, such as linezolid,
quinupristin-dalfopristin, daptomycin, telavancin, new
glycopeptides, ceftaroline, and ceftobiprole, have been introduced
or are under clinical development (Aksoy D Y and S Unal (2008) Clin
Microbiol Infect 14:411-420). As an option, current antibiotics to
which strains such as MRSA are resistant may be resurrected as
viable candidates in the treatment of MRSA when used in combination
with other agents, offering a new dimension of potential
anti-infectives. The application and use of lysin in combination
with antibiotic has potential to circumvent bacterial resistance by
virtue of the very low probability of development of resistance to
the lysin component.
[0187] In order to more fully evaluate PlySs2's applicability to
clinical Staphylococcal infections, time kill studies were
undertaken against numerous Staphylococcus aureus strains,
including methicillin resistant and methicillin sensitive strains.
Time kill assays were performed according to CLSI methodology (CLSI
document M07-A9 column 32 No. 2) on 42 methicillin resistant S.
aureus (MRSA) strains and 20 methicillin sensitive S. aureus (MSSA)
strains. Cultures of each strain
(5.5.times.10.sup.5-1.times.10.sup.6 starting inoculum) were
treated with PlySs2 lysin and with antibiotics daptomycin,
oxacillin or vancomycin for comparison for 6 hours with aeration.
MRSA and MSSA strains were treated with PlySs2, daptomycin and
vancomycin. MSSA strains were also treated with oxacillin.
1.times.MIC concentrations of the different antibiotics were
utilized, based on published and established antibiotic MIC values.
PlySs2 lysin treatment was at approximately 1.times.MIC as
previously determined (see TABLE 2 above). Culture aliquots were
removed hourly up to 6 hours (time points taken at 15 and 30 min,
lhr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr) and added to a PBS/charcoal
solution (to inactivate each drug), which was then serially diluted
and plated for bacterial viability. The resulting log CFU/mL was
plotted for each culture. Growth controls were included for each
strain and represent bacterial growth in the absence of any
antibacterial agent. Exemplary log kill curves for selected MRSA
strains are depicted in FIG. 1. Exemplary log kill curves for
selected MSSA strains are depicted in FIG. 2. A summary plot of the
time kill studies with the MRSA and MSSA strains is shown in FIG.
3.
[0188] A listing of strains used in the studies provided herein,
including cross-reference for recognized and available strain names
is provided below in TABLE 6.
TABLE-US-00008 TABLE 6 Strain List Laboratory Designation Strain
Common Strain (CFS #) Type Designation* 223 MRSA BAA-1720 241 MRSA
NRS100 243 MSSA NRS107 245 MRSA NRS070 250 MSSA NRS149 253 MSSA
NRS155 254 MSSA NRS156 263 MRSA NRS387 269 MRSA NRS123 (MW2) 270
MRSA NRS383 553 MRSA ATCC 43300 554 MSSA ATCC 25923 581 MSSA ATCC
29213 650 MRSA 052C 738 MRSA NRS192 743 MRSA NRS255 832 MRSA NRS671
926 MRSA BK20781 927 MRSA W15 *NARSA ("NRS") and ATCC ("ATCC" and
"BAA") strain designations are indicated where appropriate.
Additional names reflect strain designations available in the
literature.
[0189] PlySs2 has Rapid Kill Kinetics In Vitro
[0190] Rapid-kill kinetics are desirable in the clinical setting to
treat patients with fulminant bacterial infections. To test the
rate of antimicrobial activity in vitro, we used time-kill assays
(Mueller M et al (2004) Antimicrob Agents Chemotherapy 48:369-377)
in which 1.times.MIC drug concentrations were tested across 20
different MSSA and 42 MRSA strains. PlySs2 reached bactericidal
levels (Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically. Vol. 32 (Wayne (Pa.): Clinical and
Laboratory Standards Institute (US), 2012) (.gtoreq.3-log.sub.10
reduction in CFUs) within 30 minutes (FIGS. 4A and B). In contrast,
daptomycin required 6 hours to reach bactericidal levels while
vancomycin and oxacillin achieved only 2-log.sub.10 kill within 6
hours. Rapid-kill kinetics were also obtained for PlyS s2 against
sets of 15 different contemporary MSSA (FIG. 4C) or MRSA (FIG. 4D)
isolates, illustrating the efficient bactericidal activity of
PlySs2 on relevant clinical isolates. The potent activity of PlySs2
was further illustrated by electron microscopy showing extensive
bacteriolysis of S. aureus cocci after only 15 seconds of
treatment; the speed of PlySs2 action is consistent with a
bactericidal effect immediately upon contact (FIG. 4E).
[0191] All MRSA and MSSA strains tested are killed rapidly with
PlySs2, with maximal kill (ie, .gtoreq.3 log reduction) observed
generally within the first hour of incubation with lysin and logs
reduced to 1 log CFU/ml (the effective lower limit of the test) in
most instances. Daptomycin or vancomycin reduce growth of most MRSA
and MSSA strains by 2-3 logs observed generally over a few hours or
more of incubation, with daptomycin being the most effective
against most strains. Oxacillin was the least effective of the
antibiotic agents against MSSA strains. In all instances, PlySs2
kill was greater and faster than any antibiotic.
[0192] The studies were expanded to include testing of various S.
aureus strains, including vancomycin intermediate sensitive S.
aureus (VISA), vancomycin resistant S. aureus (VRSA), linezolid
resistant S. aureus (LRSA) and daptomycin resistant S. aureus
(DRSA), with PlySs2 lysin, daptomycin, vancomycin and linezolid,
using methods as described above. A tabulation of studies
undertaken with MSSA, MRSA, VISA, VRSA, LRSA and DRSA strains of
Staphylococcus aureus is provided in TABLE 2 above with minimal
inhibitory concentrations (MICs) of PlySs2 and various antibodies
provided for various strains.
Example 2
[0193] While PlySs2 lysin alone kills more rapidly than antibiotics
alone, as shown above, no information regarding the capability or
effectiveness of PlySs2 in combination with antibiotics is known or
available. Bacterial kill studies were undertaken to assess
combinations of PlySs2 lysin with antibiotic against Staphylococcus
aureus in vitro.
[0194] Time kill assays were performed as described above on
several MRSA strains with addition of PlySs2 or antibiotic alone or
in combination at various concentrations. Cultures of each strain
(5.5.times.10.sup.5-1.times.10.sup.6 starting inoculum) were
treated with PlySs2 lysin, antibiotic (daptomycin or vancomycin),
or combinations of PlySs2 and antibiotic for 6 hours with aeration.
In each instance, sub-MIC doses of PlySs2 and of antibiotic were
utilized in order to observe synergy and enhanced combination agent
effectiveness. Growth controls were included for each strain and
represent bacterial growth in the absence of any antibacterial
agent. Time kill curves of two MRSA strains with addition of 1/2
MIC of PlySs2 and 1/4 MIC of vancomycin are shown in FIG. 5. At
these sub-MIC doses, vancomycin or PlySs2 are ineffective or poorly
effective alone up to 6 hours. Combinations of 1/2 MIC of PlySs2
and 1/4 MIC of vancomycin result in up to 4 logs of kill of MRSA in
culture within 6 hours.
[0195] Log Kill curves of two MRSA strains with addition of 1/4 MIC
of PlySs2 and 1/8 MIC of daptomycin are shown in FIG. 6.
Combinations of 1/4 MIC of PlySs2 and 1/8 MIC of daptomycin result
in approximately 4 logs of kill of MRSA in culture within 6 hours.
FIG. 7 depicts another combination study based on 1.times.MIC
values of PlySs2 and daptomycin on MRSA strain 650 (052C Tomasz
strain--1.times.10.sup.9 starting inoculum). PlySs2 lysin is added
at16 daptomycin is added at 1 .mu.g/ml. While each single agent
alone results in 4-5 log kill at the added concentrations, the
combination of PlyS s2 and daptomycin provides complete kill (log
kill to the detection limit of the assay) within 2 hours.
Example 3
[0196] Combination therapy is particularly effective when drugs act
synergistically (Cottarel G & Wierzbowski J (2007) Trends
Biotechnology 25:547-555). Synergy assessment between PlySs2 and
cell envelope-active antibiotics was performed by the time-kill
assay, a preferred method for examining synergistic antimicrobial
activity in vitro (Mueller M et al (2004) Antimicrob Agents and
Chemotherapy 48:369-377; Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically, Vol. 32
(Wayne (Pa.): Clinical and Laboratory Standards Institute (US),
2012). Synergy studies were additionally evaluated with antibiotic
oxacillin, which is a member of the penicillin family and kills
bacteria in a distinct manner versus either of vancomycin or
daptomycin. The results of oxacillin studies either alone or in the
presence of lysin PlySs2 are shown in FIG. 8. Time-kill curves were
generated using sub-MIC concentrations of PlySs2 daptomycin,
vancomycin, and oxacillin either alone or in combinations against
clinical MRSA (FIG. 8C-8F) or MSSA (FIG. 8A-8B) isolates. Synergy
was defined as a .gtoreq.2-log 10 decrease in CFU/mL at the 6 hour
time-point for the combination compared to the most active
single-agent. Based on this criteria, PlySs2 acted synergistically
with all antibiotics evaluated against all representative MRSA and
MSSA strains evaluated (see FIGS. 4-8). An expanded set of isolates
were similarly examined and synergy was observed in 45 of 49
analyses for MSSA and 24 of 26 for MRSA with PlySs2 combined with
distinct antibiotics, including oxacillin, vancomycin and
daptomycin (TABLES 7-11 provided below).
TABLE-US-00009 TABLE 7 Synergy Time-Kill Results with Oxacillin
(MSSA) Optimal Synergistic Concentrations.sup.2 [PlySs2]
[Oxacillin] .DELTA.Log.sub.10 Inter- Strains.sup.1 .mu.g/mL
(xMIC.sup.5) .mu.g/mL (xMIC) CFU/mL.sup.3 action.sup.4 ATCC 25923
4.0 (0.13) 0.06 (0.5) 2.9 Synergy ATCC 29213 1.0 (0.13) 0.13 (0.5)
2.3 Synergy JMI 1259 1.0 (0.13) 0.13 (0.5) 2.6 Synergy JMI 1787 0.5
(0.06) 0.13 (0.5) 4.0 Synergy JMI 6408 1.0 (0.13) 0.10 (0.4) 3.3
Synergy JMI 6686 0.5 (0.06) 0.13 (0.5) 4.0 Synergy JMI 7140 0.5
(0.13) 0.50 (0.5) 4.5 Synergy JMI 8928 1.0 (0.13) 0.19 (0.4) 2.9
Synergy JMI 9365 0.3 (0.06) 0.13 (0.5) 2.5 Synergy JMI 11146 0.3
(0.03) 0.13 (0.5) 2.9 Synergy JMI 13734 0.5 (0.13) 0.10 (0.4) 4.0
Synergy JMI 13736 0.5 (0.13) 0.10 (0.4) 0.8 Additive JMI 15395 1.0
(0.06) 0.13 (0.5) 3.3 Synergy JMI 16140 2.0 (0.13) 0.10 (0.4) 3.0
Synergy JMI 33611 0.5 (0.06) 0.25 (0.5) 3.7 Synergy JMI 40979 0.5
(0.13) 0.25 (0.5) 3.0 Synergy
Legend for TABLES 7-11:
[0197] .sup.1 ATCC quality control strains and JMI isolate numbers
are shown. [0198] .sup.2 Concentrations of PlySs2 and antibiotic
used in synergy time-kill experiments. Values were carefully
determined in range-finding studies and represent concentrations
that most closely approach ideal levels of PlySs2 (that is,
resulting in a .about.2-log.sub.10 decrease in viability compared
to growth control) and antibiotic (that is, resulting in <1 log
decrease in viability compared to growth control). [0199] .sup.3
Decreases in log.sub.10 colony counts (or .DELTA. Log.sub.10
CFU/mL) are shown for cultures treated for 6 hours with drug
combination, compared to cultures treated with the most active
single agent. [0200] .sup.4 Synergy is defined by the CLSI.sup.21
as a .gtoreq.2-log.sub.10 decrease in CFU/mL. Additive interactions
are defined as a <2-log.sub.10 decrease in CFU/mL. [0201] .sup.5
xMIC, denotes percentage of MIC represented by each concentration.
For example, an xMIC value of 0.5 means that the optimal
synergistic concentration for a particular drug is 1/2 the specific
MIC value of a particular isolate or strain. The xMIC value is,
therefore, the concentration of drug used in synergy time-kill
assay divided by the MIC for that drug against the specific strain
in the absence of reductant. Key: .DELTA. Log.sub.10 CFU/mL=change
in log.sub.10 colony-forming units; MIC=minimum inhibitory
concentration.
TABLE-US-00010 [0201] TABLE 8 Synergy Time-Kill Results with
Vancomycin (MSSA) Optimal Synergistic Concentrations.sup.2 PlySs2
Vancomycin .DELTA.Log.sub.10 Inter- Strains.sup.1 .mu.g/mL
(xMIC.sup.5) .mu.g/mL (xMIC) CFU/mL.sup.3 action.sup.4 ATCC 29213
1.0 (0.03) 0.5 (0.5) 2.3 Synergy JMI 1259 1.0 (0.13) 0.5 (0.5) 3.5
Synergy JMI 1787 1.0 (0.13) 0.5 (0.5) 3.1 Synergy JMI 6408 0.5
(0.06) 0.5 (0.5) 2.8 Synergy JMI 6686 1.0 (0.13) 0.5 (0.5) 5.0
Synergy JMI 7140 0.5 (0.13) 0.5 (0.5) 3.3 Synergy JMI 8928 0.5
(0.06) 0.5 (0.5) 1.8 Additive JMI 9365 0.5 (0.13) 0.5 (0.5) 2.6
Synergy JMI 11146 0.5 (0.06) 0.3 (0.5) 3.3 Synergy JMI 13734 0.5
(0.13) 0.5 (0.5) 4.3 Synergy JMI 13736 0.5 (0.13) 0.3 (0.3) 2.3
Synergy JMI 15395 0.5 (0.03) 0.5 (0.5) 3.0 Synergy JMI 16140 1.0
(0.06) 0.5 (0.5) 3.3 Synergy JMI 18219 0.5 (0.13) 0.5 (0.5) 3.4
Synergy JMI 33611 0.5 (0.06) 0.5 (0.5) 3.6 Synergy JMI 40979 0.5
(0.13) 0.5 (0.5) 3.8 Synergy
TABLE-US-00011 TABLE 9 Synergy Time-Kill Results with Daptomycin
(MSSA) Optimal Synergistic Concentrations.sup.2 PlySs2 Daptomycin
.DELTA.Log.sub.10 Inter- Strains.sup.1 .mu.g/mL (xMIC.sup.5)
.mu.g/mL (xMIC) CFU/mL.sup.3 action.sup.4 ATCC 25923 0.5 (0.02)
0.25 (0.50) 3.1 Synergy ATCC 29213 0.3 (0.03) 0.25 (0.50) 4.3
Synergy JMI 1259 1.0 (0.13) 0.13 (0.25) 3.5 Synergy JMI 1787 0.5
(0.06) 0.07 (0.14) 3.0 Synergy JMI 6408 0.5 (0.06) 0.13 (0.25) 2.7
Synergy JMI 6686 1.0 (0.13) 0.14 (0.28) 3.4 Synergy JMI 7140 0.5
(0.13) 0.13 (0.25) 2.6 Synergy JMI 8928 0.5 (0.06) 0.13 (0.25) 2.9
Synergy JMI 9365 0.3 (0.06) 0.07 (0.28) 1.8 Additive JMI 11146 0.5
(0.06) 0.13 (0.50) 4.2 Synergy JMI 13734 0.3 (0.06) 0.08 (0.17) 3.2
Synergy JMI 13736 0.5 (0.13) 0.19 (0.38) 4.5 Synergy JMI 15395 1.0
(0.06) 0.25 (0.50) 3.2 Synergy JMI 16140 0.5 (0.03) 0.13 (0.50) 1.9
Additive JMI 18219 0.5 (0.13) 0.13 (0.25) 3.7 Synergy JMI 33611 0.3
(0.03) 0.13 (0.25) 3.4 Synergy JMI 40979 0.3 (0.06) 0.08 (0.16) 2.5
Synergy
TABLE-US-00012 TABLE 10 Synergy Time-Kill Results with Vancomycin
(MRSA) Optimal Synergistic Concentrations.sup.2 PlySs2 Vancomycin
.DELTA.Log.sub.10 Inter- Strains.sup.1 .mu.g/mL (xMIC.sup.5)
.mu.g/mL (xMIC) CFU/mL.sup.3 action.sup.4 ATCC 43300 1.0 (0.13) 0.5
(0.5) 2.1 Synergy JMI 2290 0.5 (0.06) 0.5 (0.5) 2.3 Synergy JMI
3345 0.5 (0.13) 0.5 (0.5) 5.0 Synergy JMI 4564 0.5 (0.03) 0.5 (0.5)
2.7 Synergy JMI 4789 1.0 (0.13) 0.5 (0.5) 3.3 Synergy JMI 5506 0.5
(0.13) 0.3 (0.5) 3.1 Synergy JMI 5675 0.5 (0.13) 0.5 (0.5) 3.2
Synergy JMI 6546 0.3 (0.03) 0.5 (0.5) 2.6 Synergy JMI 8941 0.5
(0.03) 0.5 (0.5) 1.8 Additive JMI 12568 0.5 (0.13) 0.5 (0.5) 5.0
Synergy JMI 18233 0.5 (0.06) 0.5 (0.5) 3.2 Synergy JMI 37753 0.5
(0.13) 0.5 (0.5) 1.6 Additive JMI 39086 0.5 (0.13) 0.3 (0.5) 2.5
Synergy
TABLE-US-00013 TABLE 11 Synergy Time-Kill Results with Daptomycin
(MRSA) Optimal Synergistic Concentrations.sup.2 PlySs2 Daptomycin
.DELTA.Log.sub.10 Inter- Strains.sup.1 .mu.g/mL (xMIC.sup.5)
.mu.g/mL (xMIC) CFU/mL.sup.3 action.sup.4 ATCC 43300 0.5 (0.06)
0.13 (0.25) 4.1 Synergy JMI 2290 1.0 (0.13) 0.25 (0.25) 4.1 Synergy
JMI 3345 1.0 (0.25) 0.25 (0.50) 5.0 Synergy JMI 4564 0.5 (0.03)
0.13 (0.25) 2.8 Synergy JMI 4789 1.0 (0.13) 0.13 (0.25) 2.4 Synergy
JMI 5506 0.5 (0.13) 0.13 (0.25) 2.3 Synergy JMI 5675 0.5 (0.13)
0.13 (0.25) 3.4 Synergy JMI 6546 1.0 (0.13) 0.06 (0.13) 3.3 Synergy
JMI 8941 0.5 (0.06) 0.13 (0.25) 2.1 Synergy JMI 12568 0.5 (0.13)
0.13 (0.50) 3.6 Synergy JMI 18233 1.0 (0.13) 0.25 (0.25) 5.7
Synergy JMI 37753 0.5 (0.06) 0.13 (0.25) 2.3 Synergy JMI 39086 0.5
(0.13) 0.13 (0.25) 3.5 Synergy
Example 4
[0202] Broth microdilution MIC testing was performed using 96 well
panels according to the methods described above for Example 1 (CLSI
methodology, CLSI document M07-A9, column 32 no 2). In the present
studies, however, both PlySs2 lysin and antibiotic daptomycin are
present together in each well at different starting concentrations.
Studies were completed with 12 different MRSA S. aureus strains. In
each instance, the MIC of PlySs2 for the strain was first
determined. DAP MICs for each strain were based on broth
microdilution MIC testing according to published methodology and
confirmed with published and available data for the tested
isolates. 5.5.times.10.sup.5-1.times.10.sup.6 cells were added to
each well and grown in the presence of various amounts of PlySs2
lysin and daptomycin for 24 hours at 37.degree. C. without
aeration. MIC values were determined by eye and confirmed by
plating bacteria to determine viable cell counts in each well of a
96-well plate. Cultures were assessed in the presence and absence
of a reducing agent (e.g., beta mercaptoethanol (BME),
dithiothreitol (DTT)). MIC values are lower relatively in the
presence of reducing agent and repeatability is improved with
reducing agent.
[0203] Dual agent MIC determinations. The dual agent MIC assay is
derived from the standard broth microdilution method (Methods for
dilution antimicrobial susceptibility tests for bacteria that grow
aerobically (2012) Vol. 32 (Clinical and Laboratory Standards
Institute (US), Wayne (Pa.)). Whereas the MIC assay dilutes one
drug across the x-axis, the MIC combo assays dilutes two drugs
together across the x axis. Two to four 96-well polypropylene
microtiter plates (Becton, Dickenson, and Company) are combined to
yield desired dilution schemes. PlySs2 is first diluted two-fold
vertically downward in column 1, yielding a concentration range of
2,048 to 1 .mu.g/mL. Daptomycin is next added at a constant
concentration (4 .mu.g/mL) to each well of column 1 All of the
wells of column 1, therefore, containing a dilution range of PlySs2
against a background of 4 .mu.g/mL daptomycin. Column 1 is next
diluted two-fold across the entire x-axis such that all the wells
of column 11 have a daptomycin concentration of 0.0037 .mu.g/mL.
After drug dilutions, cells are added (5.times.10.sup.5 CFUs/mL)
and after 24 hours of incubation at 35.degree. C. in ambient air
the MICs was recorded as the most dilute drug concentrations
inhibiting bacterial growth.
[0204] Exemplary results with eight MRSA strains are depicted in
FIGS. 9-18. In each of FIGS. 9-18, a twelve-well doubling dilution
range proceeds to the right for each panel row. Each panel
represents the well of a 96-well plate. The indicted bacterial
strains were then inoculated into each well and incubated for 24
hours at which time growth was examined Unshaded panels indicate
wells in which drug combinations inhibited growth. The yellow
(light)-shaded panels indicate the lowest drug-combination
concentrations that inhibited growth (essentially corresponding to
the MIC). The red (dark)-shaded panels indicate bacterial growth
(in other words agent combinations that did not inhibit growth).
Studies were conducted in the presence and absence of reducing
agent. In each instance, with or without the reducing agent,
significant synergy was observed, with multi-fold reduction in
amount of both lysin and antibiotic required when both were
provided in combination. Reduction in antibiotic required was
particularly significant with added lysin.
[0205] The overall experimental results with a dozen MRSA strains
are summarized in TABLE 12 below. As shown below and depicted in
FIGS. 9-18, combining PlySs2 and antibiotic (daptomycin) together
can synergistically achieve 2-4 fold reduction in the effective MIC
of PlySs2. Remarkably, combining PlySs2 and daptomycin together can
synergistically achieve 16-256 increased sensitivity (fold
reduction) in the effective MIC of the antibiotic daptomycin.
TABLE-US-00014 TABLE 12 MRSA STRAIN PlySs2 Daptomycin MIC MIC Fold
MIC MIC Fold alone.sup.1 combo.sup.2 reduction.sup.3 alone combo
reduction 553 2 1 2 1 0.0075 128 223 2 0.5 4 1 0.0075 128 270 8 2 4
1 0.015 64 269 8 2 4 1 0.015 64 (MW2) 241 4 2 2 1 0.0037 256 263 8
2 4 1 0.0075 128 650 8 4 2 1 0.015 64 827 8 4 2 1 0.0075 128 828 8
2 4 1 0.0075 128 829 8 4 2 1 0.0075 128 830 4 2 2 1 0.015 64 833 8
2 4 1 0.0075 128 .sup.1The "MIC alone" value is the single-agent
MIC (in .mu.g/mL) for each drug. .sup.2The "MIC combo" value is the
most dilute concentration of each agent (in .mu.g/mL) that, when
combined, inhibited growth. .sup.3Fold Reduction (Increased
sensitivity) corresponds to the MIC combo/MIC alone for each
agent.
Example 5
[0206] Further assessment of synergy was undertaken by performing
checkerboard assays and calculating fractional inhibitory
concentration index (FICI) values (Tallarida R J (2012) J Pharmacol
and Exper Therapeutics 342:2-8). These studies were performed using
exemplary antibiotics daptomycin, vancomycin and oxacillin. Using
this assessment, synergy is defined as inhibitory activity greater
than would be predicted by adding the two drugs together (FICI of
<0.5). Representative isobolograms for the different antibiotics
against MRSA and MSSA strains are provided in FIG. 19. Synergy was
observed for 79% (daptomycin), 86% (vancomycin), and 100%
(oxacillin) of the 29 MSSA strains and for 89% (daptomycin) and 69%
(vancomycin) of the 26 MRSA strains. The results are tabulated
below in TABLES 13-15.
TABLE-US-00015 TABLE 13 Checkerboard Analyses of PlySs2 Combined
with Oxacillin, Vancomycin, or Daptomycin (MSSA) Oxacillin
Vancomycin Daptomycin Strains FIC.sub.min Interaction FIC.sub.min
Interaction FIC.sub.min Interaction ATCC 25923 0.156 Synergy 0.500
Synergy 0.500 Synergy ATCC 29213 0.250 Synergy 0.500 Synergy 0.750
Additive JMI 243 0.187 Synergy 0.375 Synergy 0.312 Synergy JMI 332
0.187 Synergy 0.281 Synergy 0.312 Synergy JMI 1104 0.375 Synergy
0.375 Synergy 0.265 Synergy JMI 1259 0.187 Synergy 0.500 Synergy
0.500 Synergy JMI 1282 0.375 Synergy 1.00 Additive 0.750 Additive
JMI 1787 0.375 Synergy 0.500 Synergy 0.562 Additive JMI 3521 0.250
Synergy 0.75 Additive 0.375 Synergy JMI 3671 0.312 Synergy 0.562
Additive 0.500 Synergy JMI 4811 0.375 Synergy 0.375 Synergy 0.500
Synergy JMI 6408 0.375 Synergy 0.375 Synergy 0.312 Synergy JMI 6414
0.375 Synergy 0.375 Synergy 0.375 Synergy JMI 6544 0.250 Synergy
0.500 Synergy 0.375 Synergy JMI 6686 0.281 Synergy 0.500 Synergy
0.375 Synergy JMI 7140 0.375 Synergy 0.375 Synergy 0.500 Synergy
JMI 8928 0.375 Synergy 0.375 Synergy 0.500 Synergy JMI 9365 0.125
Synergy 0.375 Synergy 0.500 Synergy JMI 11146 0.250 Synergy 0.375
Synergy 0.375 Synergy JMI 13734 0.375 Synergy 0.500 Synergy 0.375
Synergy JMI 13736 0.281 Synergy 0.500 Synergy 0.500 Synergy JMI
15395 0.312 Synergy 0.250 Synergy 0.500 Synergy JMI 16195 0.375
Synergy 0.375 Synergy 0.281 Synergy JMI 16140 0.375 Synergy 0.375
Synergy 0.531 Additive JMI 18219 0.500 Synergy 0.500 Synergy 0.562
Additive JMI 24368 0.375 Synergy 0.375 Synergy 0.375 Synergy JMI
29793 0.375 Synergy 0.56 Additive 0.562 Additive JMI 33611 0.312
Synergy 0.375 Synergy 0.375 Synergy JMI 40979 0.312 Synergy 0.375
Synergy 0.312 Synergy Key: FIC.sub.min = minimum fractional
inhibitory concentration
TABLE-US-00016 TABLE 14 Checkerboard Analyses of PlySs2 Combined
with Vancomycin or Daptomycin (MRSA) Vancomycin Daptomycin Strains
FIC.sub.min Interaction FIC.sub.min Interaction ATCC 43300 0.500
Synergy 0.5 Synergy JMI 1225 0.625 Additive 0.375 Synergy JMI 1280
0.375 Synergy 0.375 Synergy JMI 2290 0.500 Synergy 0.500 Synergy
JMI 3345 0.375 Synergy 0.375 Synergy JMI 3346 0.375 Synergy 0.375
Synergy JMI 4564 0.375 Synergy 0.500 Synergy JMI 4789 0.750
Additive 0.560 Additive JMI 5506 0.562 Additive 0.375 Synergy JMI
5675 0.375 Synergy 0.375 Synergy JMI 6378 0.500 Synergy 0.560
Additive JMI 6182 1.060 Additive 0.500 Synergy JMI 6546 0.375
Synergy 0.375 Synergy JMI 7053 0.375 Synergy 0.500 Synergy JMI 8941
0.375 Synergy 0.500 Synergy JMI 9328 0.562 Additive 0.375 Synergy
JMI 10339 0.531 Additive 0.500 Synergy JMI 11127 0.531 Additive
0.625 Additive JMI 12568 0.250 Synergy 0.375 Synergy JMI 15992
0.187 Synergy 0.500 Synergy JMI 18233 0.375 Synergy 0.500 Synergy
JMI 37753 0.375 Synergy 0.375 Synergy JMI 39086 0.500 Synergy 0.500
Synergy JMI 39848 0.500 Synergy 0.375 Synergy JMI 43255 0.375
Synergy 0.375 Synergy JMI 44465 0.625 Additive 0.500 Synergy Key:
FIC.sub.min = minimum fractional inhibitory concentration.
TABLE-US-00017 TABLE 15 Summary of PlySs2Interactions with
Antimicrobial Agents Based on Checkerboard Assays and Calculated
FIC Values % Interactions with PlySs2.sup.a) Species Oxacillin
Vancomycin Daptomycin (N) Synergistic Additive Synergistic Additive
Synergistic Additive MSSA 100 0 86.2 13.8 79.3 20.7 (29) MRSA NA NA
69.2 30.8 88.5 11.5 (26) Key: FIC = fractional inhibitory
concentration; NA = not applicable.
[0207] In the checkerboard assay, drug interactions are defined as
either synergistic, additive, or antagonistic based on the
FIC.sub.min for each combination. The FIC for a drug is defined as
the MIC of the drug in combination divided by the MIC of the drug
used alone. The FIC.sub.min is based on the sum of FICs for each
drug. If the FIG. is <0.5, the combination is interpreted as
being synergistic; between >0.5 and .ltoreq.2 as additive; and
>2 as antagonistic.
Example 6
PlySs2 Accelerates Antibiotic Binding to the Cell Envelope
[0208] As a complement to the synergy studies, daptomycin and
vancomycin cell envelope-binding was examined using
BODIPY-fluorescein-labeled antibiotics in the presence and absence
of sub-MIC levels of CF-301. A time-course analysis of daptomycin
binding (FIG. 20A) shows antibiotic penetration after only 15
minutes in the presence of CF-301 (at 1/32.sup.nd MIC) versus 3
hours without CF-301. Similarly, cell wall-labeling with vancomycin
occurs within 5 minutes in the presence of CF-301 (1/8.sup.th MIC)
versus 30 minutes without CF-301 (FIG. 20B). For both antibiotics,
the labeling was first observed at bacterial division planes.
Example 7
[0209] Daptomycin binds avidly to pulmonary surfactant and
therefore is not effective in treatment of staphylococcal
pneumonia. In view of the effectiveness of PlySs2 and daptomycin in
combination against susceptible bacteria, a shown above, PlySs2
lysin and daptomycin were evaluated alone and in combination in the
presence of a commercially available surfactant, to mimic pulmonary
surfactant.
[0210] MRSA strain MW2 and MSSA strain ATCC 29213 were used in
these studies. Daptomycin and PlySs2 lysin were first evaluated
alone in the presence of surfactant (Survanta, Abbott
Laboratories). The MICs of daptomycin and PlySs2 for each strain
were determined in the presence of Survanta using broth
microdilution methods. Doubling-dilution series were established in
the presence and absence of surfactant at concentrations ranging
from 0% up to 15%. MICs were scored by eye at 24 hours and
confirmed by CFU counts in all wells. A similar study is reported
by Silverman et al., 2005 (JID, volume 191, 2149-52) using MSSA
strain 581. The fold change in MIC for daptomycin and CF301 at each
surfactant concentration (compared to MICs obtained in the absence
of surfactant) were then calculated. The fold change in MIC at
surfactant concentrations for each strain is depicted in FIG. 21.
In the presence of Survanta as surfactant, daptomycin MIC is
inhibited up to 256 fold. At a surfactant concentration of 1.25%,
daptomycin is inhibited more than 20 fold. In contrast, the MIC of
PlySs2 is inhibited 8 fold, consistently across a range from 1.25
to 15% surfactant.
[0211] The effects of combinations of PlySs2 lysin and daptomycin
were evaluated in the presence of 15% surfactant (Survanta) in a
combination MIC study. The experimental setup is similar to that
described above for the combination MIC studies without surfactant
(see Example 3). The results of synergy evaluation in the presence
of surfactant are shown in FIG. 22. Briefly, the PlySs2 lysin plus
daptomycin concentrations shown in the left-most wells were diluted
two-fold across all twelve wells of each row. MRSA strain 269 (MW2)
cells (5.5.times.10.sup.5-1.times.10.sup.6) were then added to each
well and incubated for 24 hours before growth was assessed.
Unshaded wells indicate growth inhibition. Yellow (lightly)-shaded
wells indicate the most dilute drug combinations that still inhibit
growth (ie, the MIC). Red (dark)-shaded wells indicate drug
combinations that allow growth. The PlySs2 synergy dose is 2
.mu.g/ml or 1/8 of the MIC for the strain tested. In this study,
daptomycin is effective to 0.25 .mu.g/ml, which corresponds to
1/1024 MIC of daptomycin.
Example 8
Combination Versus Single-Agent Therapy in Murine Models of
Bacteremia
[0212] Animal studies were undertaken to assess the effect of
PlySs2 in combination with antibiotic against S. aureus infection
in vivo in murine models of bacteremia. BALB/c mice were injected
IP with different levels inoculums of MRSA strains and the animals
are then dosed with drug--either antibiotic, PlySs2 lysin, or a
combination of antibiotic and PlySs2.
[0213] In a first set of studies using inoculums in the range of
10.sup.6 of bacteria, 35 ug of daptomycin was injected
subcutaneously (sc) at 5 hrs post bacterial infection, in a single
dose. This dose is equivalent to 1.75 mg/kg dose of daptomycin for
a 20 gram mouse, while thehuman equivalent dose of daptomycin in
mice would be 50 mg/kg. Dosing of 1.75 mg/kg of daptomycin in mice
is equivalent to about 3.5% of the human equivalent dose. PlySs2
was injected IP three times a day (TID), with 15 .mu.g of PlySs2
administered at 5 hrs, 9 hrs, and 13 hrs post bacterial infection
(15 .mu.g is approximately equal to a dose of 0.8 mg/kg for a 20
gram mouse). The animals are monitored and percent survival
recorded every three hours up to 24 hours post infection. Animal
survival with MRSA (strain 269 or MW2) doses of 1.8.times.10.sup.6,
1.1.times.10.sup.6, 3.0.times.10.sup.6 and 3.1.times.10.sup.6
bacteria was assessed and a compiled graph of survival data is
provided in FIG. 23 for this MRSA strain. Similar studies were
conducted with other S. aureus strains (strains 220 and 833) with
comparable results (FIGS. 24-26). In all instances, animal survival
was remarkably enhanced by combination dosing with PlySs2 lysin and
antibiotic daptomycin. These studies provide in vivo evidence of
the efficacy of combination therapy of PlySs2 lysin with antibiotic
daptomycin compared to PlySs2 lysin or antibiotic daptomycin
alone.
[0214] High-Dose Inoculum Studies
[0215] Additional animal studies in a mouse bacteremia model were
conducted to assess the ability of PlySs2 to enhance the efficacy
of standard-of-care antibiotics in vivo. In the low challenge model
(up to 7.times.10.sup.6 CFU inoculum with dosing at 4 hours),
single-agent therapy administered as a single dose of either 1.25
mg/kg PlySs2 or 2 mg/kg daptomycin resulted in 13% and 23% survival
at 72 hours, respectively (FIG. 27A). Upon combination of PlySs2
with daptomycin a significant enhancement was observed with a 72
hour survival rate of 73% (P<0.0001). The combination of PlySs2
with daptomycin was therefore superior to each agent alone under
low challenge conditions.
[0216] To test how robust the PlySs2 combination therapy may be, we
increased the bacterial inoculum to a point where human-simulated
doses of single-agent antibiotics were poorly efficacious. In this
high challenge model (10.sup.9 CFU inoculum with dosing at 2
hours), human-simulated doses of either daptomycin (50 mg/kg once
daily).sup.26 or vancomycin (110 mg/kg twice daily).sup.27 as
single agents yielded 24 hour survival rates of 47% and 20%,
respectively, and 72 hour survival rates of 31% and 3%,
respectively (FIGS. 27B and 27E). PlySs2 administered as a single
agent similarly yielded survival rates of 56/60% and 18/3% at 24
and 72 hours, respectively. In contrast, PlySs2 in combination with
either daptomycin or vancomycin achieved 24 hour survival rates of
87% and 93% at 24 hours and 82% and 67% at 72 hours, respectively,
demonstrating superiority of the combination therapies over
single-agent regimens under these challenging infection conditions.
Additional PlySs2/daptomycin combination experiments were performed
with two additional MRSA strains, yielding similar results (FIGS.
27C and 27D). When PlySs2 was further tested in combination with
oxacillin using a MSSA strain as the inoculum, the combination
treatment was again superior to that of the single agents (FIG.
27F). Taken together, the results demonstrate that
PlySs2/antibiotic combinations are more efficacious than
single-agent regimens for treating bacteremia and that
statistically significant results are obtained across various
standard-of-care antibiotics and across multiple S. aureus strains
(P<0.0001 in all cases).
[0217] Lysin PlySs2 Demonstrates Dose-Responsive, Rapid-Kill
Kinetics In-Vivo
[0218] In a murine bacteremia model, PlySs2 exhibits a clear
dose-response with survival enhancement over that observed for the
mock-injected control with as little as 0.25 mg/kg and significant
protection at the 5 mg/kg dose (data not shown). To assess the
speed of bactericidal activity of PlySs2 in vivo, MRSA CFUs were
measured in the blood of infected mice before and after
administration of PlySs2. Upon dosing 5.25 mg/kg PlySs2 at 2-hours
post-infection, a 0.5-log.sub.10 decrease in CFUs occurred in 15
minutes and a 2-login log decrease was observed within the 60
minutes of treatment, demonstrating the rapid bactericidal activity
of PlySs2 in the bloodstream of infected animals (data not
shown).
[0219] Murine Bacteremia Model.
[0220] Female BALB/c (inbred strain) mice, 5-7 weeks of age,
16.0-19.5 g body weight were purchased from Jackson Laboratories,
Bar Harbor, Me. and utilized in all mouse experiments. Exponential
phase bacterial inocula were generated by allowing bacterial cells
to grow to an optical density of .about.0.5 at 600 nm, harvested,
washed, and concentrated between
1.5.times.10.sup.7-2.times.10.sup.9 CFU/ml. The bacterial pellet
was suspended in an appropriate volume of 5% (w/v) mucin (Sigma Lot
# SLBD5666V or SLBD5666V) to achieve the specific inoculum and
placed on wet ice. Five hundred .mu.L
(.about.7.5.times.10.sup.6-1.times.10.sup.9 CFU) were injected i.p.
into mice. The study drug doses were weight-adjusted with the
injected volume between 160-200 .mu.L. Post-infection survival was
assessed every 3 or 6 hours for the first 24 hours, then at 48 and
72 hours. The experiments were repeated 2-3 times with each
treatment group containing between 10-20 mice. All experimental
manipulations using the infectious agent were conducted in a BSL-2
hood. Dead animals were removed upon observation of mortality.
Example 9
[0221] Serial passage experiments were conducted with MRSA strain
ATCC 700699 and MSSA strain ATCC 25923 to generate and evaluate
daptomycin resistance and PlySs2 lysin. These studies were
conducted to evaluate and determine whether daptomycin resistance
correlates with lysin resistance or sensitivity, including
particularly resistance or sensitivity to PlySs2 lysin. First,
daptomycin resistant clones were generated (with increasing MIC
values), in a step-wise manner, over 21 days of in vitro growth.
Then, the series of daptomycin resistant clones were assessed with
respect to lysin MIC values, by evaluating PlySs2 lysin MIC. The
results are depicted in FIG. 28. In the daptomycin resistant
clones, daptomycin MIC rises from 1 to 18 .mu.g/mL. PlySs2 lysin
MIC decreases from 8 to between 2-4 .mu.g/mL. These studies show
that daptomycin resistance correlates with PlySs2 lysin
sensitivity. These are the first studies to evaluate daptomycin
resistance and lysin sensitivity. Remarkably, in these conditions
resistance to daptomycin confers increased response to lysin,
providing enhanced rational for combination or serial
administration and therapy.
Example 10
[0222] Serial passage resistance studies were undertaken to assess
the ability of PlySs2 to suppress the emergence of antibiotic
resistance when used in combination with various standard-of-care
antibiotics used to treat Staphylococcus aureus infections. Methods
used to perform single-agent and combination serial passage
experiments are described in Palmer et al (Palmer et al (2011)
Antimicrobial Agents and Chemotherapy 55:3345-56) and Berti et al
(Berti et al (2012) Antimicrobial Agents and Chemotherapy
56:5046-53), respectively. Increases in the MIC values for the
antibiotic were assessed in triplicate for a MRSA Staphylococcus
aureus strain (MW2) grown either in the presence of antibiotic
alone or in the presence of antibiotic plus sub-MIC values of
PlySs2. The MIC for PlySs2 for strain MW2 is 32 ug/ml (without
DTT). Thus, sub-MIC values of 4 ug/ml (corresponding to 1/8 MIC) or
8 ug/ml (corresponding to 1/4 MIC) were chosen as the concentration
of PlySs2 for these experiments. Both daptomycin and vancomycin
were tested as exemplary antibiotics in this study.
[0223] For the daptomycin experiments, it was found that over the
course of the 30-day study, daptomycin resistance increased
significantly for all three daptomycin-only cultures (FIG. 30). In
these cultures the daptomycin MIC values increased from the
starting value of 1 ug/ml, to the three ending values of 128, 128
and 64 ug/ml --a 64 to 128-fold increase. For cultures that were
passaged in the presence of sub-MIC amounts of PlySs2 (4 ug/ml)
plus daptomycin, the daptomycin MIC values at the end of the 30
serial passage experiment were significantly lower-4, 4, and 4
ug/ml (a 4 fold increase). Therefore, sub-MIC concentrations of
PlySs2 suppressed the ability of the MRSA strain to mount
daptomycin resistance by 8 to 16 fold relative to the daptomycin
alone conditions. Resistance to daptomycin increased by only about
4 fold in the presence of PlySs2 lysin.
[0224] For the vancomycin experiments, it was found that over the
course of the 25-day study, vancomycin resistance increased
significantly for all three vancomycin-only cultures. In these
cultures the vancomycin MIC values increased from the starting
value of 1 ug/ml, to the three ending values of 16, 16 and 16 ug/ml
(a 16-fold increase). For cultures that were passaged in the
presence of sub-MIC amounts of PlySs2 (8 ug/ml) plus vancomycin,
the vancomycin MIC values at the end of the 25 day serial passage
experiment were significantly lower--4, 4, and 2 ug/ml (a 2 to 4
fold increase). Therefore, sub-MIC concentrations of PlySs2
suppressed the ability of the MRSA strain to mount vancomycin
resistance by 4 to 8 fold relative to the vancomycin alone
conditions. Resistance to daptomycin increased by only about 4 fold
in the presence of PlySs2 lysin.
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[0278] This invention may be embodied in other forms or carried out
in other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all aspects illustrate and not restrictive, the
scope of the invention being indicated by the appended Claims, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
[0279] Various references are cited throughout this Specification,
each of which is incorporated herein by reference in its entirety.
Sequence CWU 1
1
51245PRTStreptococcus suis 1Met Thr Thr Val Asn Glu Ala Leu Asn Asn
Val Arg Ala Gln Val Gly1 5 10 15Ser Gly Val Ser Val Gly Asn Gly Glu
Cys Tyr Ala Leu Ala Ser Trp 20 25 30Tyr Glu Arg Met Ile Ser Pro Asp
Ala Thr Val Gly Leu Gly Ala Gly 35 40 45Val Gly Trp Val Ser Gly Ala
Ile Gly Asp Thr Ile Ser Ala Lys Asn 50 55 60Ile Gly Ser Ser Tyr Asn
Trp Gln Ala Asn Gly Trp Thr Val Ser Thr65 70 75 80Ser Gly Pro Phe
Lys Ala Gly Gln Ile Val Thr Leu Gly Ala Thr Pro 85 90 95Gly Asn Pro
Tyr Gly His Val Val Ile Val Glu Ala Val Asp Gly Asp 100 105 110Arg
Leu Thr Ile Leu Glu Gln Asn Tyr Gly Gly Lys Arg Tyr Pro Val 115 120
125Arg Asn Tyr Tyr Ser Ala Ala Ser Tyr Arg Gln Gln Val Val His Tyr
130 135 140Ile Thr Pro Pro Gly Thr Val Ala Gln Ser Ala Pro Asn Leu
Ala Gly145 150 155 160Ser Arg Ser Tyr Arg Glu Thr Gly Thr Met Thr
Val Thr Val Asp Ala 165 170 175Leu Asn Val Arg Arg Ala Pro Asn Thr
Ser Gly Glu Ile Val Ala Val 180 185 190Tyr Lys Arg Gly Glu Ser Phe
Asp Tyr Asp Thr Val Ile Ile Asp Val 195 200 205Asn Gly Tyr Val Trp
Val Ser Tyr Ile Gly Gly Ser Gly Lys Arg Asn 210 215 220Tyr Val Ala
Thr Gly Ala Thr Lys Asp Gly Lys Arg Phe Gly Asn Ala225 230 235
240Trp Gly Thr Phe Lys 2452738DNAStreptococcus suis 2atgacaacag
taaatgaagc attaaataat gtaagagctc aggttgggtc cggtgtgtct 60gttggcaacg
gcgaatgcta cgctttggct agttggtacg agcgcatgat tagtccggat
120gcaactgtcg gacttggcgc tggtgtgggc tgggtcagcg gtgcaatcgg
cgatacaatc 180tctgccaaaa acatcggctc atcatacaac tggcaagcta
acggctggac agtttccaca 240tctggtccat ttaaagcagg tcagattgtg
acgcttgggg caacaccagg aaacccttac 300ggacatgtgg taatcgtcga
agcagtggac ggcgatagat tgactatttt ggagcaaaac 360tacggcggga
aacgttatcc cgtccgtaat tattacagcg ctgcaagcta tcgtcaacag
420gtcgtgcatt acatcacacc gcctggcacg gtcgcacagt cagcacccaa
ccttgcaggc 480tctcgttcct atcgcgagac gggcactatg actgtcacgg
tcgatgctct caatgttcgc 540agggcgccaa atacttcagg cgagattgta
gcagtataca agcgtggtga atcatttgac 600tatgatactg tcatcatcga
tgtcaatggc tatgtctggg tgtcttacat aggcggcagc 660ggcaaacgta
actacgttgc gacgggcgct accaaagacg gtaagcgttt cggcaatgct
720tggggtacat ttaaataa 7383139PRTStreptococcus suis 3Leu Asn Asn
Val Arg Ala Gln Val Gly Ser Gly Val Ser Val Gly Asn1 5 10 15Gly Glu
Cys Tyr Ala Leu Ala Ser Trp Tyr Glu Arg Met Ile Ser Pro 20 25 30Asp
Ala Thr Val Gly Leu Gly Ala Gly Val Gly Trp Val Ser Gly Ala 35 40
45Ile Gly Asp Thr Ile Ser Ala Lys Asn Ile Gly Ser Ser Tyr Asn Trp
50 55 60Gln Ala Asn Gly Trp Thr Val Ser Thr Ser Gly Pro Phe Lys Ala
Gly65 70 75 80Gln Ile Val Thr Leu Gly Ala Thr Pro Gly Asn Pro Tyr
Gly His Val 85 90 95Val Ile Val Glu Ala Val Asp Gly Asp Arg Leu Thr
Ile Leu Glu Gln 100 105 110Asn Tyr Gly Gly Lys Arg Tyr Pro Val Arg
Asn Tyr Tyr Ser Ala Ala 115 120 125Ser Tyr Arg Gln Gln Val Val His
Tyr Ile Thr 130 135467PRTStreptococcus suis 4Arg Ser Tyr Arg Glu
Thr Gly Thr Met Thr Val Thr Val Asp Ala Leu1 5 10 15Asn Val Arg Arg
Ala Pro Asn Thr Ser Gly Glu Ile Val Ala Val Tyr 20 25 30Lys Arg Gly
Glu Ser Phe Asp Tyr Asp Thr Val Ile Ile Asp Val Asn 35 40 45Gly Tyr
Val Trp Val Ser Tyr Ile Gly Gly Ser Gly Lys Arg Asn Tyr 50 55 60Val
Ala Thr655280PRTStaphylococcus aureus 5Met Glu Thr Leu Lys Gln Ala
Glu Ser Tyr Ile Lys Ser Lys Val Asn1 5 10 15Thr Gly Thr Asp Phe Asp
Gly Leu Tyr Gly Tyr Gln Cys Met Asp Leu 20 25 30Ala Val Asp Tyr Ile
Tyr His Val Thr Asp Gly Lys Ile Arg Met Trp 35 40 45Gly Asn Ala Lys
Asp Ala Ile Asn Asn Ser Phe Gly Gly Thr Ala Thr 50 55 60Val Tyr Lys
Asn Tyr Pro Ala Phe Arg Pro Lys Tyr Gly Asp Val Val65 70 75 80Val
Trp Thr Thr Gly Asn Phe Ala Thr Tyr Gly His Ile Ala Ile Val 85 90
95Thr Asn Pro Asp Pro Tyr Gly Asp Leu Gln Tyr Val Thr Val Leu Glu
100 105 110Gln Asn Trp Asn Gly Asn Gly Ile Tyr Lys Thr Glu Leu Ala
Thr Ile 115 120 125Arg Thr His Asp Tyr Thr Gly Ile Thr His Phe Ile
Arg Pro Asn Phe 130 135 140Ala Thr Glu Ser Ser Val Lys Lys Lys Asp
Thr Lys Lys Lys Pro Lys145 150 155 160Pro Ser Asn Arg Asp Gly Leu
Asn Lys Asp Lys Ile Val Tyr Asp Arg 165 170 175Thr Asn Ile Asn Tyr
Asn Met Val Leu Gln Gly Lys Ser Ala Ser Lys 180 185 190Ile Thr Val
Gly Ser Lys Ala Pro Tyr Asn Leu Lys Trp Ser Lys Gly 195 200 205Ala
Tyr Phe Asn Ala Lys Ile Asp Gly Leu Gly Ala Thr Ser Ala Thr 210 215
220Arg Tyr Gly Asp Asn Arg Thr Asn Tyr Arg Phe Asp Val Gly Gln
Ala225 230 235 240Val Tyr Ala Pro Gly Thr Leu Ile Tyr Val Phe Glu
Ile Ile Asp Gly 245 250 255Trp Cys Arg Ile Tyr Trp Asn Asn His Asn
Glu Trp Ile Trp His Glu 260 265 270Arg Leu Ile Val Lys Glu Val Phe
275 280
* * * * *