U.S. patent application number 08/879139 was filed with the patent office on 2003-02-06 for antibacterial therapy with bacteriophage genotypically modified to delay inactivation by the host defense system.
Invention is credited to ADHYA, SANKAR L., CARLTON, RICHARD M., MERRIL, CARL R..
Application Number | 20030026785 08/879139 |
Document ID | / |
Family ID | 22834407 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030026785 |
Kind Code |
A1 |
MERRIL, CARL R. ; et
al. |
February 6, 2003 |
ANTIBACTERIAL THERAPY WITH BACTERIOPHAGE GENOTYPICALLY MODIFIED TO
DELAY INACTIVATION BY THE HOST DEFENSE SYSTEM
Abstract
The present invention is directed to bacteriophage therapy,
using methods that enable the bacteriophage to delay inactivation
by any and all parts of the host defense system (HDS) against
foreign objects that would tend to reduce the numbers of
bacteriophage and/or the efficiency of those phage at killing the
host bacteria in an infection. Disclosed is a method of producing
bacteriophage modified for anti-HDS purposes, one method being
selection by serial passaging, and the other method being genetic
engineering of a bacteriophage, so that the modified bacteriophage
will remain active in the body for longer periods of time than the
wild-type phage.
Inventors: |
MERRIL, CARL R.; (ROCKVILLE,
MD) ; CARLTON, RICHARD M.; (PORT WASHINGTON, NY)
; ADHYA, SANKAR L.; (GAITHERSBURG, MD) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN PLLC
1050 CONNECTICUT AAVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036-5339
US
|
Family ID: |
22834407 |
Appl. No.: |
08/879139 |
Filed: |
June 19, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08879139 |
Jun 19, 1997 |
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08593269 |
Jan 29, 1996 |
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5688501 |
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08593269 |
Jan 29, 1996 |
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08222956 |
Apr 5, 1994 |
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Current U.S.
Class: |
424/93.6 ;
424/199.1 |
Current CPC
Class: |
C07K 2319/00 20130101;
Y02A 50/475 20180101; C07K 14/005 20130101; C12N 2795/10364
20130101; A61P 31/04 20180101; C12N 2795/10322 20130101; C07K
14/472 20130101; C12N 7/00 20130101; A61K 35/76 20130101; C12N
2795/10332 20130101 |
Class at
Publication: |
424/93.6 ;
424/199.1 |
International
Class: |
A61K 039/12 |
Claims
We claim:
1. A method for treating an infectious disease caused by a
bacteria, in an animal, comprising: administering to an animal in
need of such treatment, a lytic or non-lytic bacteriophage that is
specific for said bacteria in a dosage effective to substantially
eliminate the bacteria, wherein said bacteriophage has a
genetically inheritable ability to delay inactivation by an
animal's host defense system.
2. The method according to claim 1, wherein said bacteria is a drug
resistant bacteria.
3. The method according to claim 1, wherein said animal is not a
mammal.
4. The method according to claim 1, wherein said animal is a
mammal.
5. The method according to claim 4, wherein said mammal is a
human.
6. The method according to claim 1, wherein said bacteriophage has
at least a 15% longer half-life than a corresponding wild-type
phage.
7. The method according to claim 1, wherein the bacteriophage is
obtained by anti-HDS selection (serial passage) of a mutagenized or
non-mutagenized bacteriophage which is able to survive in an animal
for a longer period than a corresponding wild-type
bacteriophage.
8. The method according to claim 1, wherein the bacteria is
selected from the group consisting of Mycobacteria, Staphylococci,
Vibrio, Enterobacter, Enterococci, Escherichia, Haemophilus,
Neisseria, Pseudomonas, Shigella, Serratia, Salmonella and
Streptococci, and the bacteriophage can effectively lyse the
bacteria.
9. The method according to claim 8, wherein the bacteria is
selected from the group consisting of M. tuberculosis, M.
avium-intracellulare and M. bovis.
10. The method according to claim 1, wherein the bacteriophage is
administered by way of an aerosol to an animal's lungs.
11. The method according to claim 1, wherein the bacteriophage is
administered at a dosage of about 10.sup.6 to about 10.sup.13
pfu/kg/day.
12. The method according to claim 11, wherein the bacteriophage is
administered at a dosage of about 10.sup.12 pfu/kg/day.
13. An isolated and purified bacteriophage that has a genetically
inheritable ability to delay inactivation by an animal's host
defense system.
14. The bacteriophage according to claim 13, wherein said
bacteriophage has at least a 15% longer half-life than a
corresponding wild-type phage.
15. The bacteriophage according to claim 13, wherein the
bacteriophage is obtained by anti-HDS selection of a bacteriophage
that is able to survive in an animal's body longer than the
corresponding wild-type bacteriophage.
16. The bacteriophage according to claim 13, wherein the
bacteriophage is obtained by genetic engineering of an anti-HDS
bacteriophage that is able to survive in an animal's body longer
than the corresponding wild-type bacteriophage.
17. The bacteriophage according to claim 13, wherein said phage is
specific for bacterial families selected from the group consisting
of Escherichia, Klebsiella, Shigella, Salmonella, Serratia,
Yersinia, Enterobacter, Enterococci, Haemophilus, Mycobacteria,
Neisseria, Pseudomonas, Staphylococci, Streptococci and Vibrio.
18. A method of obtaining a bacteriophage that is able to delay
inactivation by an animal's host defense system against foreign
bodies, comprising: (a) intravenously injecting a bacteriophage
into an animal; (b) obtaining serial blood samples over time and
measuring the bacteriophage present in each sample; (c) growing a
portion of a sample obtained when about 0.1% to 1% of the
bacteriophage remain in said animal, to high titer in a host
bacteria; and (d) repeating steps (a), (b) and (c) at least once,
to yield an "anti-HDS" bacteriophage that has delayed inactivation
by an animal's host defense system.
19. The method according to claim 18, wherein step (d) is repeated
until a bacteriophage is obtained which has at least a 15% longer
half-life than a corresponding wild-type phage.
20. A method of producing a bacteriophage able to delay
inactivation by an animal's host defense system, comprising
genetically engineering a bacteriophage to express molecules on its
surface coat that delay inactivation of the bacteriophage by an
animal's host defense system.
21. The method according to claim 1, wherein the bacteriophage is
obtained by genetic engineering.
22. The method according to claim 20, wherein the bacteria is
selected from the group consisting of Mycobacteria, Staphylococci,
Vibrio, Enterobacter, Enterococci, Escherichia, Haemophilus,
Neisseria, Pseudomonas, Shigella, Serratia, Salmonella and
Streptococci, and the bacteriophage can effectively lyse the
bacteria.
23. The method according to claim 22, wherein the bacteria is
selected from the group consisting of M. tuberculosis, M.
avium-intracellulare and M. bovis.
24. The method according to claim 20, wherein the bacteriophage is
administered by way of an aerosol to an animal's lungs.
25. The method according to claim 20, wherein the bacteriophage is
administered at a dosage of about 10.sup.6 to about 10.sup.13
pfu/kg/day.
26. The method according to claim 25, wherein the bacteriophage is
administered at a dosage of about 10.sup.12 pfu/kg/day.
27. A method for treating an infectious disease caused by a
bacteria, comprising administering to an animal in need of such
treatment an antibiotic and/or a chemotherapeutic agent in
combination with a bacteriophage specific for said bacteria, in a
dosage effective to substantially eliminate the bacteria, wherein
said bacteriophage has a genetically inheritable ability to delay
inactivation by the animal's host defense system.
28. A pharmaceutical composition comprising an isolated and
purified bacteriophage which has a genetically inheritable ability
to delay inactivation by an animal's host defense system, in
combination with a pharmaceutically acceptable carrier.
29. The pharmaceutical composition according to claim 28, wherein
said composition is an aerosol formulation for administration to an
animal's lungs.
30. The pharmaceutical composition according to claim 28, wherein
said bacteriophage is in lyophilized form.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of delaying the
inactivation of bacteriophages by an animal's host defense system.
One method of delaying inactivation is the use of novel
bacteriophages whose genomes have been modified. Methods useful for
modifying the bacteriophage genome include but are not limited to
selection of mutant strains by serial passage, and the creation of
new strains by genetic engineering. Such novel bacteriophages have
the ability to delay being sequestered by, engulfed by, or
otherwise inactivated by one or more of the processes of an
animal's host defense system (HDS). This novel attribute allows the
"anti-HDS modified" bacteriophage to have a longer survival time in
an animal's body than the corresponding wild-type bacteriophage,
and that in turn allows the modified phage to be more effective
than the wild-type phage at treating (or assisting in the treatment
of) a bacterial infection.
[0002] The present invention also is directed to specific methods
of using bacteriophages for treating infectious bacterial diseases.
The route of administration can be by any means including
delivering the phage by aerosol to the lungs.
BACKGROUND OF THE INVENTION
[0003] In the 1920s, shortly after the discovery of bacterial
viruses (bacteriophages), the medical community began to
extensively pursue the treatment of bacterial diseases with
bacteriophage therapy. The idea of using phage as a therapy for
infectious bacterial diseases was first proposed by d'Herelle in
1918, as a logical application of the bacteriophages' known ability
to invade and destroy bacteria. Although early reports of
bacteriophage therapy were somewhat favorable, with continued
clinical usage it became clear that this form of therapy was
inconsistent and unpredictable in its results. Disappointment with
phage as a means of therapy grew, because the great potential of
these viruses to kill bacteria in vitro was not realized in vivo.
This led to a decline in attempts to develop clinical usage of
phage therapy, and that decline accelerated once antibiotics began
to be introduced in the 1940s and 50s. From the 1960s to the
present, some researchers who adopted certain bacteriophages as a
laboratory tool and founded the field of molecular biology have
speculated as to why phage therapy failed.
[0004] Despite the general failure of phage as therapy, isolated
groups of physicians have continued to try to use these agents to
treat infectious diseases. Many of these efforts have been
concentrated in Russia and India, where the high costs of and lack
of availability of antibiotics continues to stimulate a search for
alternative therapies. See for example Vogovazova et al.,
"Effectiveness of Klebsiella pneumoniae Bacteriophage in the
Treatment of Experimental Klebsiella Infection", Zhurnal
Mikrobiologii, Epidemiologii Immunobiologii, pp. 5-8 (April, 1991);
and Vogovazova et al., "Immunological Properties and Therapeutic
Effectiveness of Preparations of Klebsiella Bacteriophages",
Zhurnal Mikrobiologii, Epidemiologii Immunobiologii, pp. 30-33
(March, 1992)]. These articles are similar to most of the studies
of phage therapy, including the first reports by d'Herelle, in that
they lack many of the controls required by researchers who
investigate anti-infectious therapies. In addition, these studies
often have little or no quantification of clinical results. For
example, in the second of the two Russian articles cited above, the
Results section concerning Klebsiella phage therapy states that
"Its use was effective in . . . ozena (38 patients), suppuration of
the nasal sinus (5 patients) and of the middle ear (4 patients) . .
. In all cases a positive clinical effect was achieved without side
effects from the administration of the preparation". Unfortunately,
there were no placebo controls or antibiotic controls, and no
criteria were given for "improvement".
[0005] Another clinical use of phage that was developed in the
1950s and is currently still employed albeit to a limited extent,
is the use of phage lysate, specifically staphphage lysate (SPL).
The researchers in this field claim that a nonspecific,
cell-mediated immune response to staph endotoxin is an integral and
essential part of the claimed efficacy of the SPL. [See, eg., Esber
et al., J. Immunopharmacol., Vol. 3, No. 1, pp. 79-92 (1981); Aoki
et al., Augmenting Agents in Cancer Therapy (Raven, N.Y.), pp.
101-112 (1981); and Mudd et al., Ann. NY Acad. Sci., Vol. 236, pp.
244-251 (1974).] In this treatment, it seems that the purpose of
using the phage is to lyse the bacteria specifically to obtain
bacterial antigens, in a manner that those authors find
preferential to lysing by sonication or other physical/chemical
means. Here again, some difficulties arise in assessing these
reports in the literature, because, in general, there are no
placebo controls and no standard antibiotic controls against which
to measure the reported efficacy of the SPL. More significantly,
there is no suggestion in these articles to use phage per se in the
treatment of bacterial diseases. Moreover, the articles do not
suggest that phage should be modified in any manner that would
delay the capture/sequestration of phage by the host defense
system.
[0006] Since many patients will recover spontaneously from
infections, studies must have carefully designed controls and
explicit criteria to confirm that a new agent is effective. The
lack of quantification and of controls in most of the phage reports
from d'Herelle on makes it difficult if not impossible to determine
if the phage therapies have had any beneficial effect.
[0007] As there are numerous reports of attempts at phage therapy,
one would assume that had it been effective, it would have
flourished in the period before antibiotics were introduced. But
phage therapy has been virtually abandoned, except for the isolated
pockets mentioned above.
[0008] As noted above, some of the founders of molecular biology
who pioneered the use of specific phages to investigate the
molecular basis of life processes have speculated as to why phage
therapy was not effective. For example, G. Stent in his book
Molecular Biology of Bacterial Viruses, W H Freeman & Co.
(1963) pp. 8-9, stated the following:
[0009] "Just why bacteriophages, so virulent in their antibiotic
action in vitro, proved to be so impotent in vivo, has never been
adequately explained. Possibly the immediate antibody response of
the patient against the phage protein upon hypodermic injection,
the sensitivity of the phage to inactivation by gastric juices upon
oral administration, and the facility with which bacteria acquire
immunity or sport resistance against phage, all militated against
the success of phage therapy."
[0010] In 1973, one of the present inventors, Dr. Carl Merril,
discovered along with his coworkers that phage lambda, administered
by various routes (per os, IV, IM, IP) to germ-free, non-immune
mice, was cleared out of the blood stream very rapidly by the
organs of the reticulo-endothelial system, such as the spleen,
liver and bone marrow. [See Geier, Trigg and Merril. "Fate of
Bacteriophage Lambda in Non-Immune, Germ-Free Mice", Nature, 246,
pp. 221-222 (1973).] These observations led Dr. Merril and his
coworkers to suggest (in that same Nature article cited above)
over-coming the problem by flooding the body with colloidal
particles, so that the reticulo-endothelial system would be so
overwhelmed engulfing the particles that the phage might escape
capture. Dr. Merril and his coworkers did not pursue that approach
at the time as there was very little demand for an alternative
antibacterial treatment such as phage therapy in the 1970s, given
the numerous and efficacious antibiotics available.
[0011] Subsequently, however, numerous bacterial pathogens of great
importance to mankind have become multi-drug resistant (MDR), and
these MDR strains have spread rapidly around the world. As a
result, hundreds of thousands of people now die each year from
infections that could have been successfully treated by antibiotics
just 4-5 years ago. [See e.g. C. Kunin, "Resistance to
Antimicrobial Drugs--A Worldwide Calamity", Annals of Internal
Medicine, 1993;118:557-561; and H. Neu, "The Crisis in Antibiotic
Resistance", Science 257, Aug. 21, 1992, pp. 1064-73.] In the case
of MDR tuberculosis, e.g., immunocompromised as well as
non-immunocompromised patients in our era are dying within the
first month or so after the onset of symptoms, despite the use of
as many as 11 different antibiotics.
[0012] Medical authorities have described multi-drug resistance not
just for TB, but for a wide variety of other infections as well.
Some infectious disease experts have termed this situation a
"global crisis". A search is underway for alternative modes and
novel mechanisms for treating these MDR bacterial infections.
[0013] Bacteriophage therapy offers one possible alternative
treatment. Learning from the failure of bacteriophage therapy in
the past, the present inventors have discovered effective ways to
overcome the major obstacles that were the cause of that
failure.
[0014] One object of the present invention is to develop novel
bacteriophages which are able to delay inactivation by an animal's
host defense system, any component of which may be diminishing the
numbers or the efficacy of the phage that have been
administered.
[0015] Another object of the present invention is to develop a
method for treating bacterial infectious diseases in an animal by
administering to the animal an effective amount of the novel
bacteriophage, and by an appropriate route of administration.
SUMMARY OF THE INVENTION
[0016] In the present invention, novel bacteriophages are developed
by serial passage or by genetic engineering, to obtain
bacteriophages capable of delaying inactivation by any component of
an animal's host defense system (HDS) against foreign bodies. This
allows the novel phages to survive for longer periods of time in
the circulation and the tissues than the wild-type phage, thereby
prolonging viability and making these modified phages more
efficient at reaching and invading the bacteria at the site of an
infection.
[0017] The administration of an anti-HDS phage that has been
developed by serial passage or by genetic engineering will enable
the animal recipient to efficaciously fight an infection with the
corresponding bacterial pathogen. The phage therapy of this
invention will therefore be useful either as an adjunct to standard
anti-infective therapies, or as a stand-alone therapy.
[0018] The phages of the present invention can be administered by
any route, such as oral, pulmonary (by aerosol or by other
respiratory device for respiratory tract infections), nasal, IV,
IP, per vagina, per rectum, intra-ocular, by lumbar puncture,
intrathecal, and by burr hole or craniotomy if need be for direct
insertion onto the meninges (e.g. in a heavily thickened and
rapidly fatal tuberculous meningitis).
DETAILED DESCRIPTION OF THE INVENTION
[0019] One of the major obstacles to bacteriophage therapy is the
fact that when phages are administered to animals, they are rapidly
eliminated by the animal's HDS. That suggests that the phages are
not viable in the animal's circulation or tissues for a long enough
time to reach the site of infection and invade the bacteria. Thus,
the object of the present invention is to develop bacteriophages
that are able to delay inactivation by the HDS. This will prolong
phage viability in the body.
[0020] The term "host defense system" as used herein refers to all
of the various structures and functions that help an animal to
eliminate foreign bodies. These defenses include but are not
limited to the formed cells of the immune system and the humoral
components of the immune system, those humoral components including
such substances as complement, lysozymes and beta-lysin. In
addition, the organs of what has often been referred to as the
"reticulo-endothelial system" (spleen, liver, bone marrow, lymph
glands, etc.) also serve as part of the host defense system. In
addition to all the phenomena cited just above which take place
within this "reticulo-endothelial system", there has also been
described a sequestering action wherein foreign materials
(specifically including bacteriophage) are captured
non-phagocytically and non-destructively in the spleen by what is
known as the Schweigger-Seidel capillary sheaths--a phenomenon that
may or may not involve antigen capture [See e.g. Nagy, Z., Horrath,
E., and Urban, Z., Nature New Biology, 242: p. 241 (1973).]
[0021] The phrase "substantially eliminate" as used regarding the
present invention, indicates that the number of bacteria is reduced
to a number which can be completely eliminated by the animal's
defense system or by using conventional antibacterial
therapies.
[0022] Enabling bacteriophages to delay inactivation by those host
defense systems--whichever components of it may or may not be
employed in any given case--would be likely to result in an
increased in vivo killing of bacterial pathogens that are in the
host range for those bacteriophages.
[0023] In one embodiment, bacteriophages are selected by serial
passage. These will by their nature have a delay in their
inactivation by the HDS. Essentially, the serial passage is
accomplished by administering the phage to an animal and obtaining
serial blood samples over an extended period of time. Eventually
one obtains viable phage that are able to delay inactivation by the
HDS. When a period is reached where in blood samples there remains
0.01%-1.0%, and preferably 0.1%, of the number of phages originally
placed in circulation, a sample of this remaining phage is grown up
to sufficiently high titer to be injected into a second animal of
the same species. [For methods of clonal purification, see M.
Adams, Bacteriophages, Interscience Publishers, pp. 454-460
(1959)]. Serial blood samples are again obtained over time, and the
process described above is repeated iteratively so that each time
when approximately 0.1% of the phages are left, it takes longer and
longer with each serial passage to reach that point when only 0.1%
of the phage administered still remain in circulation. By this
method of clonal purification and selection, a phage strain will be
isolated that can survive at least 15% longer in the body than the
longest-surviving wild-type phage.
[0024] After a number of serial passages of these non-mutagenized
or mutagenized (see below) bacteriophage, a prototype "anti-HDS
modified" bacteriophage is obtained. As used herein, an "anti-HDS
modified" phage is defined as any phage (whether modified by serial
passage or by genetic engineering) that has a half-life within the
animal that is at least 15% greater than the half-life of the
original wild-type phage from which it was derived. Half-life
refers to the point in time when out of an initial IV dose (e.g.
1.times.10.sup.12) of a given phage, half (1.times.10.sup.6) of
them still remain in circulation, as determined by serial pfu
experiments ("pfu" meaning plaque forming units, a convenient
measure of how many phage are present in a given sample being
assayed). A 15% longer half-life indicates a successful delay of
inactivation by the HDS.
[0025] The evidence that the HDS-evading phages do in fact remain
viable for a longer period of time in the body is obtained by
demonstrating not only by the longer time that they remain in the
circulation, but also by the higher numbers of them that remain in
the circulation at a given point in time. This slower rate of
clearance is demonstrated by the fact that ten minutes after the IV
injection of 1.times.10.sup.12 of the phages into a test animal,
the number of the phages still in circulation (as measured by pfu
assays) is at least 10% higher than the number of the corresponding
wild-type phage still in circulation in the control animal, at that
point in time.
[0026] Instead of awaiting the spontaneous mutations that are
selected for in the above method, alternatively mutations can be
provoked during the growth of the phage in its host bacteria. The
mutations may produce specimens of phage that, after selection by
serial passage, are even more efficient than the non-mutagenized
phage at delaying inactivation by the host defense system.
Mutagenization is achieved by subjecting the phage to various
stimuli, such as, but not limited to, acridine compounds, ethidium
bromide in the presence of light, radioactive phosphorus, and
various forms of radiation (X-rays, UV light, etc.). Mutants
resulting from the iterative procedure described above, and that
are found to have a longer survival time than the wild-type phage,
are grown to high titer and are used to treat infectious diseases
in animals and in humans.
[0027] The phage obtained by the above methods are referred to as
"anti-HDS selected".
[0028] An altogether different method to achieve the desired result
is to genetically engineer a phage so that it expresses molecules
on its surface coat, where said molecules antagonize, inactivate,
or in some other manner impede those actions of the HDS that would
otherwise reduce the viability of the administered phages. One of
the ways to accomplish this is to engineer a phage to express
molecules that antagonize one or more of the complement
components.
[0029] Complement components fix to bacteriophages, and these
bacteriophages then adhere to certain white blood cells (such as
macrophages) that express complement receptors. Numerous peptides
have been synthesized that antagonize the functions of the various
complement components. [See e.g. Lambris, J. D. et al, "Use of
synthetic peptides in exploring and modifying complement
reactivities" in Activators and Inhibitors of Complement, ed. R.
Sim, Kluwer Academic Publishers, Boston, 1993.] Lambris et al.
(op.cit.) cite "a series of synthetic peptides spanning the
covertase cleavage site in C3 (that are) found to inhibit
complement activation by both the classical and alternative
pathways". Among the peptides cited is a six amino acid peptide
(LARSNL, residues 746-751 of C3) that "inhibits both pathways
equally well".
[0030] In one method of genetically engineering such a phage, a
fusion protein is obtained, wherein the peptide will be bound to
the carboxyl end of the surface protein of interest [See e.g.
Sambrook, J., Fritsch, E., and Maniatis, T.: Molecular Cloning. A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989]. This construct is made by cloning
the gene for the phage surface protein into a plasmid vector
system, and then cloning the oligonucleotide for the peptide of
interest into this carrying vector by in-frame fusion at the 3'-end
of the gene for the surface protein. This fusion of the gene for
the phage surface protein with the oligonucleotide for the
complement-antagonizing peptide would then be incorporated into the
phage of interest by the in vivo generalized recombination system
in the host bacteria for the phage of interest. Phage whose genomic
sequence is already completely known, and phage whose genomic
sequence is unknown or partially unknown can be used in the present
invention.
[0031] The surface expression of a recombinant
complement-antagonizing peptide is but one example of several
complement-related strategies that might be used for these
purposes. Another example would be the expression of a human
complement-antagonizing protein on the surface of a phage. Several
transplantation research facilities are currently expressing such
human complement-antagonizing proteins in transgenic animals, in
the hopes that when these transgenic organs are donated they will
not be immunologically rejected by a human recipient. [See e.g.
Genetic Engineering News, Oct. 15, 1993, p.1.] In an analogous
manner, the expression of such recombinant human
complement-antagonizing proteins on the surface of a bacteriophage
may allow the phage to delay being inactivated by the host defense
system.
[0032] In addition to complement-related strategies, there are many
other categories of molecules that can be recombinantly engineered
into a phage to delay inactivation by the host's defense system.
Other categories of molecules that could be expressed on the
surface of bacteriophages, and would fall under the scope of this
invention, include but are not limited to: interleukins and other
cytokines; autocrines; and inhibitors of the various cellular
activating or inhibiting factors (e.g. inhibitors of macrophage
activating factor). Genes for these proteins (or for active
subunits of them) can be incorporated into a phage genome so that
they will be expressed on the surface.
[0033] In addition, if it were possible to get a given bacterial
host strain to glycosylate a recombinant protein, then the purpose
of the invention could be served by introducing genes that will
express glycosylated proteins. Such proteins are known by their
negative charge to repel immune cells, such as the macrophage.
Examples might include but would not be limited to (1) the
C-terminal portion of the .beta.-subunit of human chorionic
gonadotrophin (hCG), and (2) the various glycophorins on the
surfaces of blood cells.
[0034] Phage modified in this manner are referred to as "anti-HDS
engineered".
[0035] The present invention can be applied across the spectrum of
bacterial diseases, either by serial passage of phages (mutagenized
or non-mutagenized) or by genetically engineering phages, so that
phages are developed that are specific for each of the bacterial
strains of interest. In that way, a full array of anti-HDS selected
and/or anti-HDS engineered bacteriophage is developed for virtually
all the bacterial (and other applicable) pathogens for man, his
pets, livestock and zoo animals (whether mammal, avian, or
pisciculture). Phage therapy will then be available:
[0036] 1) as an adjunct to or as a replacement for those
antibiotics and/or chemotherapeutic drugs that are no longer
functioning in a bacteriostatic or bactericidal manner due to the
development of multi-drug resistance;
[0037] 2) as a treatment for those patients who are allergic to the
antibiotics and/or chemotherapeutic drugs that would otherwise be
indicated; and
[0038] 3) as a treatment that has fewer side effects than many of
the antibiotics and/or chemotherapeutic drugs that would otherwise
be indicated for a given infection.
[0039] The second embodiment of the present invention is the
development of methods to treat bacterial infections in animals
through phage therapy with the anti-HDS modified bacteriophages
described above. Hundreds of bacteriophages and the bacterial
species they infect are known in the art. The present invention is
not limited to a specific bacteriophage or a specific bacteria.
Rather, the present invention can be utilized to develop anti-HDS
modified bacteriophages which can be used to treat any and all
infections caused by their host bacteria.
[0040] While it is contemplated that the present invention can be
used to treat any bacterial infection in an animal, it is
particularly contemplated that the methods described herein will be
very useful as a therapy (adjunctive or stand-alone) in infections
caused by drug-resistant bacteria. Experts report [See e.g.
Gibbons, A., "Exploring New Strategies to Fight Drug-Resistant
Microbes, Science, Aug. 21, 1993, pp. 1036-38.] that at the present
time, the drug-resistant bacterial species and strains listed below
represent the greatest threat to mankind:
[0041] 1. All of the clinically important members of the family
Enterobacteriaceae, most notably but not limited to the
following:
[0042] a) All the clinically important strains of Escherichia, most
notably E. coli. One among a number of candidate wild-type phages
against these particular pathogens that could be used as a starting
point for the serial passage and/or the genetic engineering of the
present invention would be ATCC phage #23723-B2. [Note: For
purposes of brevity, in all the following examples of pathogens,
the corresponding wild-type phage will be indicated by the
following phraseology: "Example of corresponding
phage:______".]
[0043] b) All the clinically important strains of Klebsiella, most
notably K. pneumoniae [Example of corresponding phage: ATCC phage
#23356-B1 ].
[0044] c) All the clinically important strains of Shigella, most
notably S. dysenteriae [Example of corresponding phage: ATCC phage
#11456a-B1].
[0045] d) All the clinically important strains of Salmonella,
including S. abortus-equi [Example of corresponding phage: ATCC
phage #9842-B1], S. typhi [Example of corresponding phage: ATCC
phage #19937-B1], S. typhimurium [Example of corresponding phage:
ATCC phage #19585-B1], S. newport [Example of corresponding phage:
ATCC phage #27869-B1], S. paratyphi-A [Example of corresponding
phage: ATCC phage #12176-B1], S. paratyphi-B [Example of
corresponding phage: ATCC phage #19940-B1], S. potsdam [Example of
corresponding phage: ATCC phage #25957-B2], and S. pollurum
[Example of corresponding phage: ATCC phage #19945-B1].
[0046] e) All the clinically important strains of Serratia, most
notably S. marcescens [Example of corresponding phage: ATCC phage
#14764-B1].
[0047] f) All the clinically important strains of Yersinia, most
notably Y. pestis [Example of corresponding phage: ATCC phage
#11953-B1].
[0048] g) All the clinically important strains of Enterobacter,
most notably E. cloacae [Example of corresponding phage: ATCC phage
#23355-B1].
[0049] 2. All the clinically important Enterococci, most notably E.
faecalis [Example of corresponding phage: ATCC phage #19948-B1]and
E. faecium [Example of corresponding phage: ATCC phage
#19953-B1].
[0050] 3. All the clinically important Haemophilus strains, most
notably H. influenzae [a corresponding phage is not available from
ATCC for this pathogen, but several can be obtained from WHO or
other labs that make them available publicly].
[0051] 4. All the clinically important Mycobacteria, most notably
M. tuberculosis [Example of corresponding phage: ATCC phage
#25618-B1], M. avium-intracellulare, M. bovis, and M. leprae.
[Corresponding phage for these pathogens are available commercially
from WHO, via The National Institute of Public Healthy &
Environmental Protection, Bilthoven, The Netherlands].
[0052] 5. Neisseria gonorrhoeae and N. meningitidis [Corresponding
phage for both can be obtained publicly from WHO or other
sources].
[0053] 6. All the clinically important Pseudomonads, most notably
P. aeuruginosa [Example of corresponding phage: ATCC phage
#14203-B1].
[0054] 7. All the clinically important Staphylococci, most notably
S. aureus [Example of corresponding phage: ATCC phage #27690-B1]
and S. epidermidis [Corresponding phage are available publicly
through the WHO, via the Colindale Institute in London].
[0055] 8. All the clinically important Streptococci, most notably
S. pneumoniae [Corresponding phage can be obtained publicly from
WHO or other sources].
[0056] 9. Vibrio cholera [Example of corresponding phage: ATCC
phage #14100-B1].
[0057] There are additional bacterial pathogens too numerous to
mention that, while not currently in the state of
antibiotic-resistance crisis, nevertheless make excellent
candidates for treatment with anti-HDS modified bacteriophages that
are able to delay inactivation by the HDS, in accordance with the
present invention. Thus, all bacterial infections caused by
bacteria for which there is a corresponding phage can be treated
using the present invention.
[0058] Any phage strain capable of doing direct or indirect harm to
a bacteria (or other pathogen) is contemplated as useful in the
present invention. Thus, phages that are lytic, phages that are
lysogenic but can later become lytic, and nonlytic phages that can
deliver a product that will be harmful to the bacteria are all
useful in the present invention.
[0059] The animals to be treated by the methods of the present
invention include but are not limited to man, his domestic pets,
livestock, pisciculture, and the animals in zoos and aquatic parks
(such as whales and dolphins).
[0060] The anti-HDS modified bacteriophage of the present invention
can be used as a stand-alone therapy or as an adjunctive therapy
for the treatment of bacterial infections. Numerous antimicrobial
agents (including antibiotics and chemotherapeutic agents) are
known in the art which would be useful in combination with anti-HDS
modified bacteriophage for treating bacterial infections. Examples
of suitable antimicrobial agents and the bacterial infections which
can be treated with the specified antimicrobial agents are listed
below. However, the present invention is not limited to the
antimicrobial agents listed below as one skilled in the art could
easily determine other antimicrobial agents useful in combination
with anti-HDS modified bacteriophage.
1 Pathogen Antimicrobial or antimicrobial group E. coli
uncomplicated urinary trimethoprim-sulfamethoxazole tract infection
(abbrev. TMO-SMO), or ampicillin; 1st generation cephalosporins,
ciprofloxacin systemic infection ampicillin, or a 3rd generation
cephalosprorin; aminoglycosides, aztreonam, or a penicillin + a
pencillinase inhibitor Klebsiella pneumoniae 1st generation
cephalosporins; 3rd gener. cephalosporins, cefotaxime, moxalactam,
amikacin, chloramphenicol Shigella (various) ciprofloxacin;
TMO-SMO, ampicillin, chloramphenicol Salmonella: S. typhi
chloramphenicol; ampicillin or TMO-SMO non-typhi species
ampicillin; chloramphenicol, TMO-SMO, ciprofloxacin Yersinia pestis
streptomycin; tetracycline, chloramphenicol Enterobacter cloacae
3rd generation cephalosporins, gentamicin, or tobramycin;
carbenicillin, amikacin, aztreonam, imipenem Hemophilus influenzae:
meningitis chloramphenicol or 3rd generation cephalosporins;
ampicillin other H. infl. infections ampicillin; TMO-SMO, cefaclor,
cefuroxime, ciprofloxacin Mycobacterium tuberculosis isoniazid
(INH) + rifampin or and M. avium-intracellulare rifabutin, the
above given along with pyrazinamide +/or ethambutol Neisseria: N.
meningitidis penicillin G; chloramphenicol, or a sulfonamide N.
gonorrhoeae: penicillin-sensitive penicillin G; spectinomycin,
ceftriaxone penicillin-resistant ceftriaxone; spectinomycin,
cefuroxime or cefoxitin, ciprofloxacin Pseudomonas aeruginosa
tobramycin or gentamycin (+/- carben- icillin, aminoglycosides);
amikacin, ceftazidime, aztreonam, imipenem Staph aureus
non-penicillinase penicillin G; 1st generation producing
cephalosporins, vancomycin, imipenem, erythromycin penicillinase
producing a penicillinase-resisting penicillin; 1st generation
cephalosporins, vanco- mycin, imipenem, erythromycin Streptococcus
pneumoniae penicillin G; 1st generation cephalosporins,
erythromycin, chloramphenicol Vibrio cholera tetracycline;
TMO-SMO
[0061] The routes of administration include but are not limited to:
oral, aerosol or other device for delivery to the lungs, nasal
spray, intravenous, intramuscular, intraperitoneal, intrathecal,
vaginal, rectal, topical, lumbar puncture, intrathecal, and direct
application to the brain and/or meninges. Excipients which can be
used as a vehicle for the delivery of the phage will be apparent to
those skilled in the art. For example, the free phage could be in
lyophilized form and be dissolved just prior to administration by
IV injection. The dosage of administration is contemplated to be in
the range of about 10.sup.6 to about 10.sup.13 pfu/per kg/per day,
and preferably about 10.sup.12 pfu/per kg/per day. The phage are
administered until successful elimination of the pathogenic
bacteria is achieved.
[0062] With respect to the aerosol administration to the lungs, the
anti-HDS modified phage is incorporated into an aerosol formulation
specifically designed for administration to the lungs by
inhalation. Many such aerosols are known in the art, and the
present invention is not limited to any particular formulation. An
example of such an aerosol is the Proventil.TM. inhaler
manufactured by Schering-Plough, the propellant of which contains
trichloromonofluoromethane, dichlorodifluoromethane and oleic acid.
The concentrations of the propellant ingredients and emulsifiers
are adjusted if necessary based on the phage being used in the
treatment. The number of phage to be administered per aerosol
treatment will be in the range of 10.sup.6 to 10.sup.13 pfu, and
preferably 10.sup.12 pfu.
[0063] The foregoing embodiments of the present invention are
further described in the following Examples. However, the present
invention is not limited by the Examples, and variations will be
apparent to those skilled in the art without departing from the
scope of the present invention. In particular, any bacteria and
phage known to infect said bacteria can be substituted in the
experiments of the following examples.
EXAMPLES
Example 1
[0064] Selection of Anti-HDS Selected Phage by Serial Passage
Through Mice.
[0065] Part 1. A stock of mutagenized or non-mutagenized lambda
coliphage strain is injected in one bolus into the blood of
laboratory mice at 10.sup.12 pfu/per kg, suspended in 0.5 cc of
sterile normal saline. The mice are periodically bled to follow the
survival of the phage in the body. The phage are assayed by plating
them on their laboratory host, E. coli. When the titer of phage in
the mice reaches a range of 0.01%-1.0%, and preferably 0.1%, of the
titer initially injected, the phage isolated at this point in time
are plaque isolated and the procedure repeated. The repeated
passage of the lambda phage between animal and bacteria yields a
phage strain that has a longer survival time in the body of the
mice. The anti-HDS selected phage strain is then subjected to
clonal (plaque) purification.
[0066] Where the phage being administered for serial passage have
first been mutagenized, the mutagenization is carried out according
to procedures known in the art [See e.g., Adams, M. Bacteriophages.
NY: Wiley Interscience, 1959, pp. 310-318 and pp. 518-520.] For
mutagenization by ultraviolet radiation, during the last 40%-90%
(and preferably 65%) of the latent period, the phage (inside the
infected host bacteria) are exposed to 3,000-6,000 ergs (and
preferably 4,500 ergs) of ultraviolet radiation per square mm. For
mutagenization by X-radiation, a wavelength of 0.95 .ANG. is used
at doses from 10-250 (and preferably 150) kiloroentgens.
Example 2
[0067] Determination that HDS Inactivation is Delayed for the
Anti-HDS Selected Phage as Compared to Wild-Type Phage.
[0068] Two groups of mice are injected with phage as specified
below:
[0069] Group 1: The experimental group receives an IV injection
consisting of 1.times.10.sup.12 of the anti-HDS selected phage,
suspended in 0.5 cc of normal sterile saline.
[0070] Group 2: The control group receives a IV injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the serially-passaged phage were derived, suspended in 0.5 cc of
sterile normal saline.
[0071] Both groups of mice are bled at regular intervals, and the
blood samples assayed for phage content (by pfu assays) to
determine the following:
[0072] 1) Assays for half-lives of the two phages: For each group
of mice, the point in time is noted at which there remains in
circulation only half (i.e., 1.times.10.sup.6) the amount of phage
as administered at the outset. The point in time at which half of
the anti-HDS selected phage have been eliminated from the
circulation is at least 15% longer than the corresponding point in
time at which half of the wild-type phage have been eliminated from
the circulation.
[0073] 2) Assays for absolute numbers: For each group of mice, a
sample of blood is taken at precisely 1 hour after administration
of the phage. At 1 hour post-injection, the numbers of
anti-HDS-selected phage in circulation are at least 10% higher than
the numbers of wild-type phage still in circulation.
Example 3
[0074] Determination that the Anti-HDS Selected Phage has a Greater
Capacity than Wild-Type Phage to Prevent Lethal Infections in
Mice.
[0075] Part 1. Peritonitis Model: An LD.sub.50 dosage of E. coli is
administered intraperitonally (IP) to laboratory mice. The strain
of E. coli used is known to be lysed by the coliphage strain that
is selected by Serial Passage. The treatment modality is
administered precisely 20 minutes after the bacteria are injected,
but before the onset of symptoms. The treatment modalities consist
of the following:
[0076] Group 1: The experimental group receives an IP injection
consisting of 1.times.10.sup.12 of the anti-HDS selected phage
lambda coliphage suspended in 2 cc of sterile normal saline.
[0077] Group 2: A first control group receives an IP injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the anti-HDS selected phage were developed, suspended in 2 cc of
normal sterile saline.
[0078] Group 3: A second control group receives an IP injection of
2 cc of normal sterile saline.
[0079] Evidence that treatment with the anti-HDS selected phage
prevented the development of a lethal event in the peritonitis
model is measured by using the following three criteria:
[0080] (1) Survival of the animal
[0081] (2) Bacterial counts: Samples of peritoneal fluid are
withdrawn every 1/2 hour from the three groups of infected mice,
and the rate of increase or decrease in E. coli colony counts in
the three groups is noted
[0082] (3) Phage control: Using the samples of IP fluid withdrawn
from the infected mice, the numbers of pfu of the anti-HDS selected
phage and the numbers of pfu of the wild-type phage are noted.
[0083] Part 2. Bacteremia Model:
[0084] An LD.sub.50 dosage of E. coli is administered intravenously
(IV) to laboratory mice, where the strain of E. coli used is known
to be lysed by the coliphage strain that was chosen for the serial
passage. The treatment modality (see below) is administered
precisely 20 minutes after the bacteria are injected, but before
the onset of symptoms. The treatment modalities consist of the
following:
[0085] Group 1: The experimental group receives an IV injection
consisting of 1.times.10.sup.12 of the anti-HDS selected lambda
coliphage suspended in 0.5 cc of sterile normal saline.
[0086] Group 2: A first control group receives an IV injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the Anti-HDS selected phage were developed, suspended in 0.5 cc of
normal sterile saline.
[0087] Group 3: A second control group receives an IV injection of
0.5 cc of normal sterile saline.
[0088] Evidence that treatment with the anti-HDS selected phage
prevented the development of a lethal event in the bacteremia model
is measured using the following three criteria:
[0089] (1) Survival of the animal
[0090] (2) Bacterial counts: Samples of blood are withdrawn every
1/2 hour from the three groups of infected mice, and the rate of
increase or decrease in E. coli colony counts in the three groups
is noted.
[0091] (3) Phage counts: In the samples of blood withdrawn from the
infected mice, the numbers of pfu of the anti-HDS selected phage
and the numbers of pfu of the wild-type phage are noted.
Example 4
[0092] Genetic Engineering of Phage to Express Molecules that
Antagonize the Host Defense System, thereby Enabling the Phage to
Delay Inactivation by the Host Defense System.
[0093] Part 1. Making the Fusion Protein
[0094] Step 1. A double-stranded DNA encoding the complement
antagonizing peptide LARSNL is synthesized on an automated
oligonucleotide synthesizer using standard techniques.
[0095] Step 2. The gene for the phage coat surface protein of
interest (see part 2, below) is cloned into a plasmid vector
system, by techniques known in the art. The oligonucleotide that
has been prepared in Step 1 is cloned into the plasmid vector
system by in-frame fusion at the 3'-end of the gene for the surface
protein.
[0096] Step 3. The fusion gene is then incorporated into a phage by
the in vivo generalized recombination system in the host bacteria
for the phage. The phage then expresses the fusion protein on its
surface.
[0097] Part 2: Selecting phage coat surface proteins for fusion
with the peptide/protein of interest.
[0098] A. Incorporating the Gene for the Complement-antagonizing
Peptide into a Phage whose Genome is Well Characterized
[0099] The orfX gene, which encodes a carboxy-terminal tail protein
of lambda coliphage, is one for which it is known that foreign
nucleotide sequences can be introduced without there being
disruption of the structure or function of the phage. The tail
surface protein expressed by the orfX gene is made into a fusion
protein with the complement-antagonizing peptide, by the plasmid
vector method described in part 1 above.
[0100] B. Incorporating a Gene for a Complement-antagonizing
Peptide into a Phage whose Genome is not Well Characterized.
[0101] Step 1. Selection of the phage surface protein to be fused
with the complement-antagonizing peptide:
[0102] a) Isolation of phage coat surface proteins and preparation
of antibodies thereto:
[0103] (1) Samples of the phage of interest are broken up in 0.1%
SDS detergent for 2 minutes at 95.degree. C. The mixture is cooled
and placed in 9M urea, and is then separated by high resolution 2 D
gel electrophoresis. The protein fragments are then isolated from
the gel, and processed as described below.
[0104] (2) Samples of the protein fragments from the gel are
injected into animals to produce either polyclonal or monoclonal
antibodies.
[0105] (3) Antibodies are isolated and then marked with uranium.
These marked antibodies are reacted against whole phage. The marker
pinpoints precisely those proteins on the surface of the phage to
which the antibodies have bound through visualization by
electronmicroscopy. [See e.g. K. Williams and M. Chase, ed.,
Methods In Immunology and Immunochemistry, Vol.1, 1967, Academic
Press.] Antibodies directed against a surface protein extending
outward from the surface of the virus are retained for further
use.
[0106] b) Preparation of phage restriction fragments:
[0107] The genome of the phage is cut by restriction enzymes, and
the resulting restriction fragments are cloned into expression
vector plasmids. Each of these plasmids expresses its corresponding
protein, creating a pool of expressed proteins.
[0108] c) Reacting the expressed proteins with the marked
antibodies:
[0109] The antibodies directed against a surface protein extending
outward from the surface of the virus are reacted against the
proteins expressed by the plasmid vectors.
[0110] d) Correlating coat protein antibodies to the plasmid
vectors that express the genes for those coat proteins:
[0111] The reaction of a marked antibody with an expressed protein
pinpoints the expression plasmid whose enclosed restriction
fragment expresses the particular protein. Thus, the genomic
fragment encoding each coat surface protein is determined using the
marked antibodies.
[0112] e) Determining that the gene in its entirety has been
obtained:
[0113] The restriction fragments containing a gene for a surface
protein are micro-sequenced by the Sanger technique to determine
(1) the precise amino acid sequence of the coat surface proteins;
(2) the presence of a start and a stop signal (indicating that the
gene in its entirety has been obtained); and (3) the presence of
either a C-terminal or an N-terminal amino acid.
[0114] Step 2. Fusing the candidate phage surface protein with the
complement-antagonizing peptide of interest:
[0115] a) Preparing the coat protein gene for fusion:
[0116] The gene for a surface protein is contained in its plasmid
expression vector. The oligo-nucleotide for the
complement-antagonizing peptide is spliced into this plasmid
expression vector by in-frame fusion at the 3'-end of the coat
surface protein.
[0117] b) Incorporating the fusion gene into the phage of
interest:
[0118] The fusion gene is incorporated into the phage by the in
vivo generalized recombination system in the host bacteria for the
phage.
[0119] c) Demonstrating that the phage expresses the fusion
protein:
[0120] The phage is incubated with a corresponding heavy
metal-marked antibody that has been raised against the coat surface
protein. The marker is detected on the phage by electronmicroscopy
only if the phage has expressed that fusion protein on its surface.
[See e.g. K. Williams and M. Chase, Methods In Immunology and
Immunochemistry, Vol.1, 1967, Academic Press.]
Example 5
[0121] Demonstration that the Genetically Engineered Phage Delay
Inactivation by the HDS, Compared to Wild-type Phage:
[0122] Two groups of mice are injected with phage as specified
below:
[0123] Group 1: The experimental group receives an IV injection
consisting of 1.times.10.sup.12 of the genetically modified phage,
suspended in 0.5 cc of sterile normal saline.
[0124] Group 2: The control group receives an IV injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the genetically modified phage were derived, suspended in 5 cc of
sterile normal saline.
[0125] Both groups of mice are bled at regular intervals, and the
blood samples assayed for phage content (by pfu assays) to
determine the following:
[0126] 1) Assays for half-lives of the two phages: For each group
of mice, the point in time is noted at which there remains in
circulation only half (i.e., 1.times.10.sup.6) the amount of phage
as administered at the outset. The point in time at which half of
the genetically modified phage have been eliminated from the
circulation is at least 15% longer than the corresponding point in
time at which half of the wild-type phage have been eliminated from
the circulation.
[0127] 2) Assays for absolute numbers: For each group of mice, a
sample of blood is taken at precisely 1 hour after administration
of the phage. The criterion used is that at 1 hour post-injection,
pfu assays reveal that the numbers of genetically engineered phage
still in circulation in the experimental animal are at least 10%
higher than the numbers of wild-type phage still in circulation in
the control animal.
Example 6
[0128] Determination that the Genetically Engineered Phage has a
Greater Capacity than Wild Type Phage to Prevent Lethal Infections
in Mice.
[0129] Part 1. Peritonitis Model:
[0130] An LD.sub.50 dosage of E. coli is administered
intraperitonally (IP) to laboratory mice. The strain of E. coli
used is one known to be lysed by the coliphage strain that has been
genetically engineered. The treatment modality is administered
precisely 20 minutes after the bacteria are injected, but before
the onset of symptoms. The treatment modalities consist of the
following:
[0131] Group 1: The experimental group receives an IP injection
consisting of 1.times.10.sup.12 of the genetically engineered
lambda coliphage suspended in 2 cc of sterile normal saline.
[0132] Group 2: A first control group receives an IP injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the genetically modified phage were developed, suspended in 2 cc of
sterile normal saline.
[0133] Group 3: A second control group receives an IP injection of
sterile normal saline.
[0134] Evidence that treatment with the genetically modified phage
prevented the development of a lethal event in the peritonitis
model is measured by using the following three criteria:
[0135] (1) Survival of the animal
[0136] (2) Bacterial counts: Samples of peritoneal fluid are
withdrawn every 1/2 hour from the three groups of infected mice,
and the rate of increase or decrease in E. coli colony counts in
the three groups is noted
[0137] (3) Phage control: Using the samples of IP fluid withdrawn
from the infected mice, the numbers of pfu of the genetically
engineered phage versus the numbers of pfu of the wild-type phage
are noted.
[0138] Part 2. Bacteremia Model:
[0139] An LD.sub.50 dosage of E. coli is administered intravenously
(IV) to laboratory mice, where the strain of E. coli used is known
to be lysed by the coliphage strain that was genetically
engineered. The treatment modality (see below) is administered
precisely 20 minutes after the bacteria are injected, but before
the onset of symptoms. The treatment modalities consist of the
following:
[0140] Group 1: The experimental group receives an IV injection
consisting of 1.times.10.sup.12 of the genetically engineered
lambda coliphage suspended in 0.5 cc of sterile normal saline.
[0141] Group 2: A first control group receives an IV injection
consisting of 1.times.10.sup.12 of the wild-type phage from which
the genetically engineered phage were developed, suspended in 0.5
cc of sterile normal saline.
[0142] Group 3: A second control group receives an IV injection of
0.5 cc of sterile normal saline.
[0143] Evidence that treatment with the genetically engineered
phage prevented the development of a lethal event in the bacteremia
model is measured using the following three criteria:
[0144] (1) Survival of the animal
[0145] (2) Bacterial counts: In the samples of blood that are
withdrawn every 1/2 hour from the three groups of infected mice,
the absolute numbers as well as the rate of increase or decrease in
E. coli colony counts is noted, for each of those three groups.
[0146] (3) Phage counts: In the samples of blood withdrawn from the
infected mice, the numbers of pfu of the genetically engineered
phage and the numbers of pfu of the wild-type phage are noted.
* * * * *