WO2000069269A1

WO2000069269A1 - Strains of bacteriophage useful for rescuing patients infected with vancomycin-resistant enterococcus faecium

Info

Publication number: WO2000069269A1
Application number: PCT/US2000/006718
Authority: WO
Inventors: Carl R. Merril; Richard M. Carlton; Sankar Adhya
Original assignee: Exponential Biotherapies, Inc.; National Institutes Of Helth
Priority date: 1999-05-13
Filing date: 2000-05-12
Publication date: 2000-11-23
Also published as: JP2002543816A; AU4971900A; CA2373486A1; EP1180937A1; WO2000069269A9; EP1180937A4

Abstract

The present invention involves the use of specific phages, designated ENB6 and ENB13, that kill many clinical isolates of vancomycin-resistant Enterococcus faecium and of vancomycin-sensitive Enterococcus faecium. The genome of one of the phage strains, ENB6 has been partially sequenced, and is shown to not contain nucleotide sequences for known bacterial virulence genes or for the vancomycin resistance cassette. Its efficacy in rescuing mice from otherwise-fatal bacteremias is documented herein.

Description

Strains of Bacteriophage Useful for Rescuing Patients Infected With

Vancomycin-Resistant Enterococcus Faecium

FIELD OF THE INVENTION

Several clinically important species of bacteria have become multidrug

resistant ("MDR"). One of these is Enterococcus faecium, a commensal that does

not cause disease in its habitual niche (the intestines) but which can breach the gut

barrier and cause bacteremias ifthe immune system fails to eliminate the bacteria.

Immunocompromised patients cannot eliminate these bacteria, and deaths in such

patients are becoming increasingly commonplace.

As E. faecium acquired resistance to increasing numbers of antibiotics (e.g.

penicillins, cephalosporins and aminoglycosides), treatment options became

progressively narrowed until vancomycin was the drug of last resort. In 1989 the

first clinical isolates of vancomycin-resistant E__ faecium (VREF) were reported.

Physicians were then confronted with a pathogen that was difficult and often

impossible to treat. In recent years the prevalence of these vancomycin-resistant

strains has increased to the point that hospitals typically report that approximately

40% of the E. faecium clinical isolates are vancomycin resistant. Correspondingly,

fatal bacteremias are being reported in steadily increasing numbers.

While the pharmaceutical industry does introduce new antibiotics from time

to time, it has become commonplace that new antibiotics become rapidly resisted

by multidrug resistant ("MDR") bacteria. For example, Synercid® recently entered the market as a treatment for vancomycin-resistant bacteria, including VREF.

Resistance to this antibiotic began to appear even while it was in clinical trials, and

by the time it was approved for commercial sales approximately 20% of VREF

clinical isolates were reported fully resistant to this new antibiotic. The reason that

MDR bacteria are so efficient at resisting newer antibiotics (even those to which

they have never been exposed) is that the resistance mechanisms they've acquired

enable them to defeat many different classes of antibiotics. For example, a mutant

efflux pump can transport out many classes of drugs; and a mutation in the

ribosomal subunit targeted by antibiotics can defeat several classes of drugs. An

alternative to antibiotics is therefore needed to control such MDR bacteria.

Bacte ophage (phage) therapy offers one such alternative. The

present invention describes several examples of phage strains (for example ENB6)

that rescues mice from a fulminant VREF bacteremia.

BACKGROUND OF THE INVENTION

As described in US Patent No# 5,688,501 by Merril et al (and

incorporated by reference herein), phage therapy of human bacterial infections

failed for a number of technical reasons. One of the technical reasons was that

phages tend to be rapidly cleared from the systemic circulation by the filtering action

of the organs of the reticulo-endothelial system (RES). This rapid clearance

prevents the phages from remaining in circulation long enough to reach and infect

the target bacteria infecting the patient. The above-cited invention solved the problem of rapid clearance by

introducing a novel approach called "serial passage". In that technique, a large

number of phages of a wild-type strain are injected into an animal, blood samples

are taken at various intervals, and any phage particles still remaining in circulation

at the time of the venipuncture will be present therein and can be grown to high titer

on the host bacteria. This technique therefore selects for phage variants whose

surface coat proteins are not readily detected by the RES, and such variants are

amplified by cloning at the end of each round of serial passage. Since the phages

being selected must be able to produce plaques on the lawn of the host bacteria,

the technique also selects for those mutants that retain their ability to lyse the target

bacteria. Finally, the long-circulating phage mutants obtained thereby were superior

to the wild-types from which they were derived, in terms of rescuing an animal from

an otherwise-fatal bacteremia. In the above-referenced patent, the bacterial target

was a strain of E. coli, and the wild-type phage strain used was lambda coliphage.

In the present invention, phage stains that attack VREF hosts have

been discovered by the present inventors. These strains were discovered through

screening samples of sewage from the waste management system of Montgomery

County, Maryland.

SUMMARY OF THE INVENTION

Phage strains were grown by standard techniques known in the art,

by plating them on clinical isolates of VREF which were obtained from hospitalized patients (with no identifiers as to the name of the patients). These stains are lytic

when propagated in many clinical isolates of VREF.

These phage strains were grown to high titer, and they were characterized

and defined through the methods described below using the phage strain ENB6 as

an example.

DETAILED DESCRIPTION OF THE INVENTION

Details on the characterization of and host range of phage ENB6 are

provided in this section. Details on the phage's utility, in terms of rescuing animals

from an otherwise-lethal bacteremia, are provided in the section that follows.

1. Genomic sequencing

50 mg of phage ENB6 DNA was sheared and then random fragments were

"shotgun cloned" into an M13-based vector for sequencing. The raw data was pre-

screened and then the individual sequences were compiled into overlapping

contigs.

The ENB6 genome contains at least 120 kb of DNA as determined by

sequencing and gel electrophoretic analyses of extracted DNA. A total of 94.4 kb

of nucleotide sequence has been defined at 99% confidence, while 24.7 kb has been defined at a lower level of confidence. The remaining amount is presently

undefined.

2. Analyzing the phage's genome for nucleotide sequences of interest, using

homology searches on databases as well as PCR probes

The ENB6 nucleotide sequences have been compared to all genes and

proteins registered in the databases using two alignment algorithms, BLASTN

(nucleotide sequence comparisons) and BLASTX (putative amino acid sequence

comparisons). All alignments of high confidence matched genes and gene products

of other bacteriophages including those for head, tail, polymerase and lysin

proteins. No extensive and significant match was found at the nucleotide or

predicted protein level to recognized whole genes of bacterial factors for

pathogenicity, infectivity, invasion, attachment or antibiotic resistance. However,

four short and dispersed alignments to these kinds of undesirable factors were

found as shown in Figure 1 and Table 1. The fraction of each protein exhibiting

some similarity to a potential gene product from ENB6 is not greater than 30 % in

any example, meaning, at best, only a partial gene exists. The short lengths of

identity suggest that only a subtle similarity exists at the amino acid sequence level.

If actually translated into protein products, these fragmented domains would either

be not functional or unfamiliar.

Thus, we find no evidence of whole genes for potentially hazardous factors

in the known nucleotide sequence of phage ENB6. Understanding that only part of Table 1. Undesirable proteins found by BLASTX alignments of theoretical

proteins derived from ENB6 nucleotide sequence.

the genome was screened by database searches, we have undertaken a second

approach to inspecting the ENB6 phage for potentially undesirable genes. We have

designed oligonucleotide primers for physical screening of the phage DNA by PCR

amplification. The genes searched are listed in Table 2.

Thus we have used sequence alignment searches and physical tests for

known genes to address the concern for a potential risk of horizontal gene transfer

through the therapeutic use of bacteriophage phage ENB6.

3. Electron microscopic study

Figure 2 is an electron microscopic picture of phage ENB6.

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 10³ to about 10¹³

pfu/per kg/per day, and preferably about 10¹² pfu/per kg/per day. The phage are Table 2. Proteins screened by PCR amplification of ENB6 DNA.

administered until successful elimination of the pathogenic Enterococcus

faecium is achieved.

As used in the present application, the term "substantially reduce"

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.

The present invention will be particularly useful in treating critically ill

patients or those with severe underlying disease or immunosuppression (e.g.

patients in ICUs or in oncology or transplant wards), patients who have had an

intraabdominal or cardio-thoracic surgical procedure or an indwelling urinary or

central venous catheter, and persons who have had a prolonged hospital stay or

received multi-antimicrobial and/or vancomycin therapy.

Deposits of ENB6 (ATCC # PTA-40) and ENB13 (ATCC # PTA-39) were

made on May 12, 1999 at the American Type Culture Collection, 10801

University Blvd., Manassas, VA. 20110-2209.

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.

EXAMPLES

1. VREF Bacteremia Rescue Experiment #1 : Dose-Finding Study

Figures 3 and 4 show the results of a dose-finding study.

Materials and Methods:

We had previously determined that the 2xLD₅₀ dose for a clinical VREF isolate

designated CRMEN44 is 1 x 10⁹ CFU, when injected I. P. into one month-old balb/c

female mice, in other studies (data not shown here), we had determined that the

I. P. injection of this bacteria strain causes a bacteremia within 15 minutes, and that

the I. P. injection of phage ENB6 causes a viremia within 15 minutes. In this study,

the following dosages of phage ENB6 were administered once (and only once) I. P.,

exactly V_ hour after the bacterial challenge: 3 x 10⁹, 3 x 10⁸, 3 x 10⁶, and 3 x 10⁴

PFU plaque forming units (PFU). In addition, a dose of 3 x 10⁹ PFU was

administered I. P. to another set of animals, as a control, with no bacterial challenge.

The non-parametric rating scale for observable signs of illness is as follows:

5 = Normal animal; 4 = Mild lethargy; 3 = Mild lethargy + Ruffled fur; 2 = the above,

plus exudate around the eyes; 1 = Moribund; and 0 = Dead.

Results:

Phage administered as a control did not produce any detectable symptoms in the

animals. Bacteria administered without any phage treatment caused the death of

all the animals, within 48 hours. With the two highest dosages of phage there were no deaths, and the animals recovered within 24 hours from the minimal signs of

illness that had developed, with no relapse over a period of 21 days of observation.

While there were some deaths with the two lowest dosages of phage, nevertheless

roughly half the animals in these groups survived (and recovered completely) after

becoming moderately ill.

Discussion:

Phage ENB6 rescues animals from an otherwise-fatal dose of VREF, a bacterial

pathogen for which no consistently reliable antibiotic is currently available. The

infection here is fulminant, using a concentration of bacteria (10⁹, which will be very

concentrated in the 3 ml of blood in a mouse's circulatory system) that is orders-of-

magnitude greater than that found in bacteremic humans (where titers in blood

reach only 10² to 10⁴ CFU per cc).

Conclusion:

While an IND approval will be required from the FDA before such phages can be

administered therapeutically to humans, phage strains that lyse VREF hosts in-vitro

should be able to kill the bacterial targets wherever encountered (in-vivo), whether

within the mouse or the human circulatory system. Moreover, multiple phage doses

will be employed in treating humans. In this experiment only one dose was

administered, in order to demonstrate the ability of the phages to grow exponentially

in number and to thereby overwhelm the target bacteria. 2. VREF Bacteremia Rescue Experiment #2: Delayed Treatment

Figures 5 and 6 show the results of delay in the treatment of a fulminant

bacteremia.

Materials and Methods:

Same as in Experiment 1 , except for the dosage and timing of the phage

administration. In this experiment, only the highest dose (3 x 10⁹) of phage ENB6

was administered. After the I. P. bacterial challenge, the one (and only one) I. P.

administration of the phage dose was delayed until one or another of the following

time points: 2, 5, 8, 14, 18 and 24 hours. One group of animals received no phage

treatment, as a control.

Results:

With no treatment, all animals were dead within 48 hours. With treatment delayed

2 hours and 5 hours, all animals survived (after becoming moderately ill). With

treatment delayed from 8 -24 hours approximately half the animals died, but for the

half that survived, even though the degree of illness reached was severe,

nevertheless there was full and complete recovery by day 4 or 5, with no relapse.

Discussion:

Even when treatment of a fulminant bacteremia in mice is delayed, phage ENB6

tends to rescue the animals from an otherwise-fatal dose of VREF. The rescue is 100% with delays up to and including 5 hours. With delays between 8 - 24 hours,

approximately 50% of the animals survive and go on to recover completely.

Conclusion:

While an IND approval will be required from the FDA before such phages can be

within the mouse or the human circulatory system. In the human, concentrations of

VREF are orders-of-magnitude lower than the concentrations achieved here, so it

should be that much easier to achieve a therapeutic success. Moreover, in treating

humans, multiple administration of phage will be employed. In this experiment only

one dose was administered, in order to demonstrate the ability of the phages to

grow exponentially in number and to thereby overwhelm the target bacteria.

Claims

We claim:

1. A wild-type phage which is lytic for strains of vancomycin-resistant

Enterococcus faecium (VREF) as well as for strains of vancomycin-sensitive

Enterococcus faecium (VSEF), wherein said phage is selected from the group

consisting of ENB6 (ATCC# PTA-40), and ENB13 (ATCC# PTA-39)

2. A method for treating an Enterococcus faecium infection comprising

administering an amount of a phage selected from the group consisting of ENB6

(ATCC# PTA-40), and ENB13 (ATCC# PTA-39) effective to eradicate or

substantially reduce an Enterococcus faecium infection to a patient in need of such

treatment.

3. The method according to claim 2, wherein said Enterococcus faecium is

vancomycin-resistant Enterococcus faecium.

4. The method according to claim 2, wherein said phage is administered by

a route selected from the group consisting of orally, topically, intravenously, intra-

arterially, intraperitoneally, intrathecally, by inhalation, by nasal spray, by irrigation

of a wound, by suppository, and by enema.

5. The method according to claim 2, wherein said phage is administered at

a total dose of between 10³ - 10¹² PFU.

6. The method according to claim 5, wherein said phage is administered at

a total dose of between 10⁵ - 10¹¹ PFU.

7. The method according to claim 2, further comprising administering an

antibiotic.

8. A method for reducing the probability of an Enterococcus faecium

colonization becoming an infection comprising administering an amount of phage

selected from the group consisting of ENB6 (ATCC# PTA-40), and ENB13 (ATCC#-

PTA-39) effective to reduce the probability of such colonization becoming an

infection to a patient at risk for an Enterococcus faecium infection.

9. The method according to claim 8, wherein said phage is administered by

of a wound, by suppository, and by enema.

10. The method according to claim 8, wherein said phage is administered at

a total dose of between 10³ - 10¹² PFU.

11. The method according to claim 10, wherein said phage is administered

at a total dose of between 10⁵ - 10¹¹ PFU.

12. A pharmaceutical composition comprising a phage selected from the

group consisting of ENB6 (ATCC# PTA-40), and ENB13 (ATCC# PTA-39) in

combination with a pharmaceutical carrier.

13. The composition according to claim 12, further comprising an antibiotic.