Note: Descriptions are shown in the official language in which they were submitted.
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CHIMERIC RECEPTOR BINDING PROTEINS RESISTANT TO PROTEOLYTIC
DEGRADATION
TECHNICAL FIELD
[1] The present disclosure relates to chimeric receptor binding proteins,
in particular derived
from bacteriophage receptor binding proteins, able to withstand proteolytic
digestion, in
particular gastrointestinal proteolytic digestion, bacterial delivery vehicles
comprising said
chimeric receptor binding proteins, and the use thereof in efficient transfer
of a desired payload
into a target bacterial cell population, in particular after oral
administration.
BACKGROUND
[2] One of the critical aspects to be addressed when considering protein-
based DNA delivery
vectors, such as packaged phagemids or Eligobiotics , is their stability in in
vivo conditions.
Depending on the route of administration, packaged phagemids may be exposed to
different
factors that may affect their stability and functionality. For instance,
orally administered packaged
phagemids will have to traverse the gastrointestinal tract: harsh conditions,
such as the low pH
in the stomach and the presence of certain digestive enzymes, may have a
negative effect on
the structural stability of the particles.
[3] Phages have evolved to be stable in a wide range of conditions [1].
From the evolutionary
perspective, being able to resist these conditions is a clear advantage for
any phage.
[4] However, it is well known that many phages are not resistant to low pH
values for a long
period of time [1], [2], although this can be circumvented by the use of
stomach acid neutralizers
[3]¨[6]. Similarly, some phages have evolved to be resistant to digestive
enzymes, such as those
found in pancreatic juices (trypsin, chymotrypsin, etc.), while some others
are readily degraded
[4], [7], [8] although the exact mechanisms of degradation have not been
studied in detail.
[5] From these facts, it can be concluded that for the development of a
highly successful
optimal phage-derived DNA delivery vector, such as an Eligobiotic , it is
useful to obtain a vector
which is stable in in vivo conditions.
[6] The present disclosure provides a solution to this need.
[7] A powerful engineering pipeline has been developed to generate phage-
derived DNA
delivery vectors with improved or modified host ranges as disclosed in
W02020109339. To do
this, the natural variability of phage parts has been exploited to generate
functional protein
chimeras in existing phage scaffolds: for instance, one was able to modify the
tropism and
injection efficiency of packaged phagemids by modifying the two main host
range determinants
of lambdoId phages such as the lambda phage, the gpJ and STF (Side Tail Fiber)
proteins.
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[8] In the course of vector development, it was observed that one had to
differentiate
between functionality and stability. A given protein chimera (for instance, a
STF fusion) can
exhibit an ideal functionality, for example can contribute to a high injection
efficiency into a target
strain in in vitro conditions, but may be affected when exposed to pancreatin
(i.e. still functional
but less stable). This was an unpredictable aspect of the protein engineering
process so far:
starting from two different STFs that are not degraded in the presence of
pancreatin could yield
a protein chimera that was less resistant to proteolytic digestion.
[9] Different direct (on the packaged phagemids itself) or indirect (on the
environment of the
packaged phagemids) approaches can be envisioned to protect these protein
chimera from in
vivo proteolytic digestion, e.g. a suitable formulation, such as controlled or
delayed release
formulations enabling the release of packaged phagemids displaying said
protein chimera in the
intestine or the colon. The present disclosure shows that another solution is
to act directly on
said protein chimera.
SUMMARY
[10] The present disclosure is based on the unexpected finding that, by
specifically designing
a small fusion region (also called linker region) between two different STFs,
it is possible to
render a chimeric lambda based STF protein, which was initially engineered to
be fully functional
but was less stable in the presence of pancreatin, both functional and highly
stable.
[11] It is worth noting that in natural phage STFs, proteolytically
degradable residues exist
that due to conformation or interaction with other residues/proteins may not
be accessible for
degradation in normal conditions. However, such residues may become accessible
for
degradation when these STFs are used to produce chimeras. It has been
specifically
demonstrated that introducing point mutations in phenylalanine (F) and lysine
(K) residues
present in the linker region, corresponding to a region of about 10 to 12
amino acids adjacent to
the insertion site of chimeric lambda STF-V1 0, rendered the chimeric lambda
STF-V1 0 protein
partially resistant to pancreatin hence with increased stability, while the
original chimeric lambda
STF-V1 0 protein was not resistant to pancreatin at all.
[12] It has also been demonstrated that designing the linker region to
include a short
sequence which was initially present at the N-terminus end of the C-terminal
region of V10 tail
fiber used to produce the chimera, rendered the chimeric lambda STF-V10
protein highly
resistant to pancreatin (without introducing further mutations in the linker
region).
[13] Further it has been demonstrated, in another chimeric receptor binding
protein, namely
a functional chimeric lambda STF-K5 protein, which was not very stable in the
presence of
pancreatin, that introducing, in the linker region, the same helix-forming
sequence initially
present at the N-terminus of the V10 tail fiber, rendered the chimeric STF-K5
protein highly
resistant to pancreatin hence strongly stable.
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[14] Furthermore, it has been demonstrated, in another functional chimeric
lambda STF-K5
protein, which was not very stable in the presence of pancreatin, that
introducing, in the linker
region another helix-forming sequence present within the STF protein of the
Escherichia phage
ZG49 (which has homology with the wild-type K5 protein), rendered the chimeric
STF-K5 protein
very highly resistant to pancreatin.
[15] The present disclosure thus concerns a chimeric receptor binding protein
(RBP) resistant
to proteolytic digestion, in particular within the gastrointestinal tract,
wherein said chimeric RBP
comprises a portion of a receptor binding protein derived from a bacteriophage
fused through a
designed linker region consisting of 1 up to 70 amino acids, more particularly
of 1 up to 30 amino
acids, to a portion of a receptor binding protein derived from a different
bacteriophage, wherein
said linker region is designed to be resistant to proteolytic digestion, in
particular within the
gastrointestinal tract. In a particular embodiment, said chimeric RBP is
resistant to proteolytic
digestion by pancreatin, and said linker region is designed to be resistant to
proteolytic digestion
by pancreatin.
[16] In a particular embodiment, said RBP is a side tail fiber (STF)
protein, an L-shape fiber,
a long tail fiber or a tailspike. In a particular embodiment, said chimeric
RBP comprises a portion
of a STF protein derived from a lambdoId bacteriophage fused through a
designed linker region
consisting of 1 up to 70 amino acids (more particularly of 1 up to 30 amino
acids), to a portion of
a RBP protein derived from a different bacteriophage. In a particular
embodiment, said chimeric
RBP comprises an N-terminal region of a STF protein derived from a lambdoId
bacteriophage,
fused through a designed linker region consisting of 1 up to 70 amino acids
(more particularly of
1 up to 30 amino acids), to a C-terminal region of a RBP protein derived from
a different
bacteriophage, wherein said N-terminal region and C-terminal region are fused
within a site of
the N-terminal STF region, called insertion site, having at least 80% identity
with a site selected
from the group consisting of amino acids SAGDAS (SEQ ID NO: 1), ADAKKS (SEQ ID
NO: 2),
MDETNR (SEQ ID NO: 3), SASAAA (SEQ ID NO: 4), and GAGENS (SEQ ID NO: 5). In a
particular embodiment, said insertion site has at least 80% identity with
sequence GAGENS
(SEQ ID NO: 5). In a particular embodiment, said designed linker region is at
the C-terminal end
of the insertion site. In a particular embodiment, said designed linker region
is part of the N-
terminal region or of the C-terminal region of the chimeric RBP.
[17] In a particular embodiment, at least one amino acid of the designed
linker region,
corresponding to an amino acid of the wildtype domain sequence which is likely
to be targeted
by trypsin and/or chymotrypsin, is mutated compared to the wildtype domain
sequence. In said
particular embodiment, said designed linker region may be part of the C-
terminal region of the
chimeric RBP and said at least one amino acid may be located within the 15
amino acids
following the insertion site. In still said particular embodiment, said at
least one amino acid may
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be selected from the group consisting of lysin (K), arginine (R),
phenylalanine (F), tryptophan
(W), tyrosine (Y) leucine (L) and methionine (M).
[18] In another particular embodiment, said N-terminal region or said C-
terminal region
comprises the sequence of the linker region, said sequence being identical to
the corresponding
sequence in the N-terminal region or C-terminal region of the RBP from which
it is derived, and
said sequence conferring resistance to proteolytic digestion to said chimeric
RBP compared to
the original chimeric RBP only differing by the absence of said linker region.
[19] In another particular embodiment, said designed linker region
comprises or consists of
an heterologous amino acid sequence which is not derived from the N-terminal
region or from
the C-terminal region of the chimeric RBP. In said embodiment, said designed
linker region may
comprise or consist of an amino acid sequence which is derived from a RBP
which is not one of
the RBP from which the N-terminal region and the C-terminal region of the
chimeric RBP are
derived.
[20] In a particular embodiment, said designed linker region may consist of
10 up to 20 amino
acids. In said embodiment, said designed linker region may comprise or consist
of an amino
acid sequence GSATDVMIQL (SEQ ID NO: 6) or GSATDVMIQLA (SEQ ID NO: 7). In said
embodiment, said sequence may be located directly after the insertion site.
[21] In an alternative embodiment, said designed linker region may consist
of 50 up to 65
amino acids. In said embodiment, said designed linker region may comprise or
consist of an
amino acid sequence SEQ ID NO: 34 or SEQ ID NO: 37. In said embodiment, said
sequence
may be located directly after the insertion site.
[22] In a particular embodiment, the designed linker region comprises a
helix or helical
bundle.
[23] In a particular embodiment, the N-terminal region of said STF protein
derived from a
lambdoId bacteriophage corresponds to amino acids 1 to 528 of the lambda STF
protein of
sequence SEQ ID NO: 8. In a particular embodiment, the C-terminal region of
said STF protein
derived from said different bacteriophage corresponds to amino acids 218 to
875 of the STF
protein of sequence SEQ ID NO: 16. In said embodiment, said chimeric RBP may
comprise or
consist of the sequence SEQ ID NO: 9 or SEQ ID NO: 10. In another particular
embodiment, the
C-terminal region of said STF protein derived from said different
bacteriophage corresponds to
amino acids 208 to 875 of the STF protein of sequence SEQ ID NO: 16. In said
embodiment,
said chimeric RBP may comprise or consist of the sequence SEQ ID NO: 11. In a
particular
embodiment, the C-terminal region of said STF protein derived from said
different bacteriophage
corresponds to amino acids 28 to 632 of the STF protein of sequence SEQ ID NO:
12. In said
embodiment, said chimeric RBP may comprise or consist of the sequence SEQ ID
NO: 13 or
SEQ ID NO: 14. In a particular embodiment, the C-terminal region of said STF
protein derived
from said different bacteriophage corresponds to amino acids 62 to 632 of the
STF protein of
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sequence SEQ ID NO: 12. In said embodiment, said chimeric RBP may comprise or
consist of
the sequence SEQ ID NO: 38 or SEQ ID NO: 40.
[24] The present disclosure also concerns a lambdoId bacterial delivery
vehicle for use in in
vivo delivery of a DNA payload of interest into a targeted bacterial cell,
wherein said lambdoId
delivery vehicle comprises the chimeric RBP provided herein. In a particular
embodiment, said
chimeric RBP is a chimeric STF protein as disclosed herein. In said
embodiment, said chimeric
STF protein may be a functional STF protein. In still said embodiment, the
delivery vehicle may
further comprise a functional lambdoId bacteriophage gpJ protein and/or a
functional lambdoId
bacteriophage gpH protein. In a particular embodiment, the chimeric STF
protein has enzyme
activity such as depolymerase activity and the bacterial cell population of
interest comprises
encapsulated bacteria. In a particular embodiment, one or more of the chimeric
STF protein, the
gpJ protein and/or the gpH protein are engineered to increase the efficiency
of transfer of the
DNA payload into a targeted bacterial cell population. In a particular
embodiment, the delivery
vehicle comprises the chimeric RBP comprising or consisting of the sequence
SEQ ID NO: 11
and the gpJ chimeric protein 1A2 comprising or consisting of the sequence SEQ
ID NO: 27.
[25] In a particular embodiment, the bacterial cell population is selected
from the group
consisting of E. coli bacteria, K. pneumoniae and other species of interest.
[26] In a particular embodiment, said bacterial delivery vehicle comprises
said DNA payload
of interest. In a particular embodiment, the DNA payload comprises a nucleic
acid of interest
selected from the group consisting of Cas nuclease gene, a Cas9 nuclease gene,
a guide RNA,
a CRISPR locus, a toxin gene, a gene expressing an enzyme such as a nuclease
or a kinase, a
TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane
protein, a
structural protein, a secreted protein, a gene expressing resistance to an
antibiotic or to a drug
in general, a gene expressing a toxic protein or a toxic factor, and a gene
expressing a virulence
protein or a virulence factor, and or any of their combination. In said
embodiment, the nuclease
may target cleavage of a host bacterial cell chromosome or a host bacterial
cell plasmid. In said
embodiment, the cleavage may occur in an antibiotic resistant gene. In a
particular embodiment,
the nucleic acid of interest encodes a therapeutic protein. In another
particular embodiment, the
nucleic acid of interest encodes an antisense nucleic acid molecule.
[27] The present disclosure also relates to a pharmaceutical or veterinary
composition
comprising a bacterial delivery as disclosed herein and a pharmaceutically
acceptable carrier.
In a particular embodiment, said composition is for oral administration.
[28] The present disclosure also provides a method for in vivo delivery of a
DNA payload of
interest into a subject comprising, administering to said subject the
pharmaceutical or veterinary
composition as provided herein.
[29] Another object of the disclosure relates to providing a method for
treating a disease or
disorder caused by bacteria comprising administering to a subject having a
disease or disorder
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in need of treatment a therapeutically efficient amount of a pharmaceutical or
veterinary
composition disclosed herein. In a particular embodiment, said disease or
disorder is a bacterial
infection, a metabolic disorder or a pathology involving bacteria of the human
microbiome. In still
a particular embodiment, said composition is administered orally.
[30] The present disclosure also provides pharmaceutical or veterinary
compositions for use
in a method for treating a disease or disorder caused by bacteria. In a
particular embodiment,
said disease or disorder is a bacterial infection, a metabolic disorder or a
pathology involving
bacteria of the human microbiome. In still a particular embodiment, said
composition is
administered orally.
[31] The present disclosure further concerns a method for reducing the amount
of virulent
and/or antibiotic resistant bacteria in a bacterial population comprising
contacting the bacterial
population with a bacterial delivery vehicle as provided herein. Another
object concerns
providing bacterial delivery vehicles for use in a method for reducing the
amount of virulent
and/or antibiotic resistant bacteria in a bacterial population.
BRIEF DESCRIPTION OF THE FIGURES
[32] Figure 1: Stability of lambda packaged phagemids in SIF (Simulated
Intestinal Fluid).
Left group of bars, wild-type lambda packaged phagemid produced from CYC3 in
MG1655;
central group of bars, lambda 1A2-V10 packaged phagemids in MG1656-OmpC0157;
right
group of bars, 1A2-V10 packaged phagemids on H10 (0157) strain. Y axis shows
particle titer
per pL.
[33] Figure 2: Lambda STF-V10 engineered variants. Arrows depict predicted
trypsin and
chymotrypsin sites (not all sites shown for clarity reasons)
[34] Figure 3: Stability of lambda STF-V10 variants in different
conditions. Left group of bars,
original lambda STF-V10 variant (SEQ ID NO: 15); second group of bars, STF-V10-
[FA] variant
(SEQ ID NO: 9); third group of bars, STF-V10-[AAH] variant (SEQ ID NO: 10);
fourth group of
bars, STF-V10-Helix variant (SEQ ID NO: 11). The Y axis shows CFU count per
pL.
[35] Figure 4: Shedding of lambda packaged phagemids 1A2 gpJ - STF-V10 (1A2-
V10) over
time in un-colonized mice (n=3). The dose bars on the left correspond to the
titration after
production of the packaged phagemids. "black bars": 1A2 activity; "grey bars":
V10 activity.
[36] Figure 5: Shedding of lambda packaged phagemids 1A2 - STF-V10-[FA] (n=4)
and 1A2
- STF-V10-[Helix] (n=3) at t=6h after administration in un-colonized mice.
"black circles", 1A2
activity; "white triangle", V10 activity.
[37] Figure 6. Shedding of lambda packaged phagemids 1A2 - STF-V10-[Helix]
over time
(n=5 mice) following a single oral administration of these packaged phagemids.
Legend:
H10Astx = V10 activity; MG1656-OmpC0157 = 1A2 activity.
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[38] Figure 7: Percentage of pRFP curing from H10Astx/pRFP in vivo (n=10 mice)
at three
different time points after the first dose of the cocktail (1A2 - STF-V10-[FA]
and 1A2 - STF-V10-
[Helix]) : t = 6h, black; t = 24h, light grey; t = 48h, dark grey.
[39] Figure 8: Intestinal decolonization of the STEC strain H1OWT overtime
after 5 doses of
packaged phagemids: colonization overtime of the control group gavaged with
buffer (sucrose
bicarbonate).
[40] Figure 9: Intestinal decolonization of the STEC strain H1OWT overtime
after 5 doses of
packaged phagemids: colonization overtime of the test group treated with
lambda packaged
phagemids 1A2 - STF-V10-[Helix].
[41] Figure 10: Stability of lambda packaged phagemids 1A2 - K5 in PBS. Black
bars, PBS
only; white bars, PBS plus pancreatin at pH 6.8. Left group of bars, activity
in MG1656-
OmpC0157; right group of bars, LMR 503 strain. Y axis shows particle titer per
pL.
[42] Figure 11: Stability of lambda packaged phagemids 1A2 - K5 5.0 Helix
variant. Black
bars, PBS only; white bars, PBS plus pancreatin at pH 6.8. Left group of bars,
activity in
MG1656-OmpC0157; right group of bars, LMR 503 strain. Y axis shows particle
titer per pL.
[43] Figure 12: Stability of lambda packaged phagemids 1A2 - K5 5.1 Helix
variant. Black
bars, PBS only; white bars, PBS plus pancreatin at pH 6.8. Left group of bars,
activity in
MG1656-OmpC0157; right group of bars, LMR 503 strain. Y axis shows particle
titer per pL.
[44] Figure 13: Overlay of the sedimentation coefficient distribution data
of the 3 Eligobiotics
(EB) batches analyzed by svAUC in Example 3. The integration ranges for EB
packaged with 3
or 4 copies of the payload are depicted by dotted lines.
[45] Figure 14: Relative abundance of Eligobiotics containing either 3 or
4 copies of the
payload. Absorbance signals at 260 and 280 nm for each population defined in
svAUC were
integrated and used to calculate their relative abundance in each batch of
Eligobiotics .
[46] Figure 15: Stability of lambda packaged phagemids 1A2 - K5 in PBS. Black
bars: PBS
only; white bars: PBS plus pancreatin at pH 6.8. Left group of bars: activity
in MG1656-
OmpC0157; right group of bars: LMR 503 strain. Y axis shows particle titer per
pL.
[47] Figure 16: Intestinal decolonization of the LMR 503 strain over time
after 1 dose of
Eligobiotic . Colonization over time of the test group treated with
Eligobiotic harboring the A8
gpJ, the K5 9.1 STF and the plasmid p775. D8 represents the days after
colonization of mice
with the LMR 503 strain; TO, T8 represent the time 0 (pre-treatment levels)
and 8h after
treatment with Eligobiotic .
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DETAILED DESCRIPTION
Chimeric receptor binding protein (RBP)
[48] The present disclosure relates to a chimeric receptor binding protein
(RBP) resistant to
proteolytic digestion, in particular within the gastrointestinal tract,
wherein said RBP comprises
a portion of a receptor binding protein derived from a bacteriophage fused
through a designed
linker region consisting of 1 to 70 amino acids, more particularly of 1 to 30
amino acids, to a
portion of a corresponding receptor binding protein derived from a different
bacteriophage,
wherein said linker region is designed to be resistant to proteolytic
digestion, in particular within
the gastrointestinal tract.
Resistance to proteolytic digestion
[49] By "proteolytic digestion" is meant herein proteolysis of a protein
mediated by an enzyme
having any protease activity. By "proteolytic digestion within the
gastrointestinal tract" is meant
herein proteolysis of a protein mediated by an enzyme having protease activity
in any part of the
gastrointestinal tract, such as in the mouth, the esophagus, the stomach, the
small intestine or
the large intestine. In a particular embodiment, said proteolytic digestion is
within the small
intestine. In a more particular embodiment, said proteolytic digestion is
within the duodenum.
[50] As well-known from the skilled person, proteolytic digestion within the
duodenum is
mainly affected by bile salts and pancreatin. In a particular embodiment, said
proteolytic
digestion is by pancreatin. By "pancreatin" is meant herein a mixture of
pancreatic enzymes
including trypsin and chymotrypsin, and optionally amylase and lipase. In
another particular
embodiment, said proteolytic digestion is by trypsin and/or chymotrypsin. By
"trypsin" is meant
herein an enzyme of the EC 3.4.21.4 category, which is a serine protease from
the PA clan
superfamily, found in the digestive system of many vertebrates, where it
hydrolyzes proteins.
Typically, trypsin cleaves peptides on the C-terminal side of lysine and
arginine amino acid
residues, but If a proline residue is on the carboxyl side of the cleavage
site, the cleavage may
not not occur, and if an acidic residue is on either side of the cleavage
site, the rate of hydrolysis
may be be slower. By "chymotrypsin" is meant herein an enzyme of the EC
3.4.21.1 category,
which is a serine protease from the PA clan superfamily, found in the
digestive system of
vertebrates, where it hydrolyzes proteins. Typically, chymotrypsin cleaves
peptide bonds
involving L-isomers of tyrosine, phenylalanine, and tryptophan.
[51] By "resistant to proteolytic digestion" is meant herein that the
chimeric RBP is not
cleaved by said proteases and/or remains stable when contacted with said
proteases and/or
keeps its activity when contacted with said proteases. Techniques to determine
if a protein is
resistant to proteolytic digestion by pancreatin, in particular by trypsin
and/or chymotrypsin,
typically include exposing said protein to simulated intestinal fluid (SIF) in
the presence or
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absence of pancreatin, typically at 2% w/v, for example at pH 6.8, typically
for 3 h, in particular
at 37 C, then determining the activity of said treated protein (for example by
titration of the
bacterial delivery vehicle comprising said chimeric RBP in bacteria which are
specifically
targeted by packaged phagemid comprising said RBPs) and comparing it with the
activity of
same but non-treated protein. In the context of the present disclosure, a
chimeric RBP is
preferably considered as resistant to proteolytic digestion if the titer of
the bacterial delivery
vehicle comprising said chimeric RBP in bacteria which are specifically
targeted by said RBPs
decreases of 1 log or less, after treatment with pancreatin, typically at 2%
w/v, for example at
pH 6.8, typically for 3 h, in particular at 37 C compared to the titer of the
same but non-treated
bacterial delivery vehicle comprising the same chimeric RBP targeting the same
bacteria.
Chimeric RBP
[52] As used herein, a receptor binding protein or RBP is a polypeptide that
recognizes, and
optionally binds and/or modifies or degrades a substrate located on the
bacterial outer envelope,
such as, without limitation, bacterial outer membrane, LPS, capsule, protein
receptor, channel,
structure such as the flagellum, pili, secretion system. The substrate can be,
without limitation,
any carbohydrate or modified carbohydrate, any lipid or modified lipid, any
protein or modified
protein, any amino acid sequence, and any combination thereof.
[53] Such bacteriophage RBPs, from which the RBP portions are derived,
include, for
example, "L-shape fibers", "side tail fibers (stfs)", "long tail fibers" or
"tailspikes." In a preferred
embodiment, the RBPs have a host range that is directed to specific bacterial
cells of the host
or subject microbiome. In one specific aspect, the different RBP of the
chimeric RBPis derived
from any bacteriophage or from any bacteriocin.
[54] In an embodiment, said chimeric RBP is a chimeric side tail fiber
(STF) protein.
[55] In a particular embodiment, the chimeric STF comprises an N-terminal
region of an STF
derived from a lambdoId bacteriophage, preferably a lambda or lambda-like
bacteriophage,
fused through said designed linker region, to a C-terminal region of a STF
protein derived from
a different bacteriophage. Such chimeric RBPs include those having an altered
host range
and/or biological activity such as, for example, depolymerase activity.
[56] As used herein, lambdoId bacteriophages comprise a group of related
viruses that infect
bacteria. The viruses are termed lambdoId because one of the first members to
be described
was lambda (A). LambdoId bacteriophages are members of the Caudovirus order
(also known
as tailed bacteriophages) and include those bacteriophages with similar
lifestyles, including, for
example, the ability to recombine when intercrossed, possession of identical
pairs of cohesive
ends, and prophages that are inducible by ultraviolet irradiation. Although
members of the order
may have genomes that vary at the nucleotide level, they carry regions of
sufficient nucleotide
sequence identity to guide recombination between themselves, typically giving
rise to a fully
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functional phage that has all the necessary genes. (See, for example, Casjens
and Hendrix
(2015) Virology 479-480:310-330). For purposes of the present disclosure,
lambdoId
bacteriophages for use as delivery vehicles, as well as lambdoId STF for use,
would be
understood generally by one skilled in the art.
[57] LambdoId phages can be defined as belonging to the lambda supercluster
based on
genomic analysis [9]. Within this supercluster, several clusters can be
distinguished, each having
a prototypical phage. The phage-like clusters and their members (between
brackets) are:
Lambda-like (lambda (A), HK630, HK629), phi80-like (phi80, HK225, mEp237), N15-
like (N15,
PY54, phiK02), HK97-like (HK97, HK022, HK75, HK106, HK140, HK446, HK542,
HK544,
HK633, mEpX1, mEpX2, mEp234, mEp235, mEp390, ENT39118), E518-like (E518, Oslo,
SPN3UB), Gifsy-2-like (gifsy-2, gifsy-1, Fels-1, mEp043, mEp213, OP-1639, CTD-
lo, mEp640,
FSL SP-016), BP-4795-like (BP-4795, 2851, stx2-1717, YYZ-2008), SfV-like (SfV,
Sfll, Sf IV,
Sfl, oP27, 5T64B), P22-like (P22, L, SPN900, 5T64T, 5T104, 5T160, ep5i10n34,
g341, SE1,
Emek, (p20, IME10, Sf6, HK620, CUS-3, SPC-P1), APSE-1-like (APSE-1, APSE-2),
933W-like
(933W, stx1o, stx2o-I, stx2o-II, stx2-86, min27, o24B, P13374, TL-2011c, VT2-
sakai,
VT2o 272), HK639-like (HK639), oES15-like (oES15), H52-like (H52), ENT47970-
like
(ENT47670), ZF40-like (ZF40), oEt88-like (oEt88). LambdoId phages further
encompass any
bacteriophage encoding a RBP having amino acids sequence homology of around
35% identity
for 45 amino acids or more, around 50% identify for 30 amino acids or more, or
around 90%
identity for 18 amino acids or more in one or more of three amino acids
regions ranging from
positions 1-150, 320-460, and 495-560 with reference to the lambda
bacteriophage STF
sequence SEQ ID NO: 8, independently of other amino acids sequences encoded by
said
bacteriophage.
[58] In the present disclosure a lambdoId STF protein includes, for
example, a protein
comprising or consisting of an amino acid sequence with at least 75% identity
up to an amino
acid corresponding to amino acid 130 of lambda STF (Uniprot P03764 SEQ ID NO:
8), in
particular up to amino acid 130 of said lambda STF.
[59] In one aspect, the STF protein includes a protein that comprises or
consists of an amino
acid sequence with 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with
the wild type lambda
STF protein amino acid sequence of SEQ ID NO: 8, or with any of the chimeric
STF proteins
disclosed herein.
[60] As used herein, the percent homology between two sequences is equivalent
to the
percent identity between the two sequences. The percent identity is calculated
in relation to
polymers (e.g., polynucleotide or polypeptide) whose sequences have been
aligned. The
percent identity between the two sequences is a function of the number of
identical positions
shared by the sequences (i.e., `)/0 homology = # of identical positions /total
# of positions x 100),
taking into account the number of gaps, and the length of each gap, which need
to be introduced
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for optimal alignment of the two sequences. The comparison of sequences and
determination of
percent identity between two sequences can be accomplished using a
mathematical algorithm,
as described in the non-limiting examples below.
[61] The percent identity between two amino acid sequences can be determined
using the
algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988))
which has been
incorporated into the ALIGN program (version 2.0), using a PAM120 weight
residue table, a gap
length penalty of 12 and a gap penalty of 4. In addition, the percent identity
between two amino
acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
48:444-453
(1970)) algorithm which has been incorporated into the GAP program in the GCG
software
package (available at www.gcg.com), using a BLOSUM62 matrix, a BLOSUM30 matrix
or a
PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4,
5, or 6. In a specific embodiment the BLOSUM30 matrix is used with gap open
penalty of 12 and
gap extension penalty of 4.
[62] In the context of the present disclosure, said RBP derived from a
bacteriophage (from
which is derived the N-terminal region of the chimeric RBP) is resistant to
proteolytic digestion
as defined above, and said RBP derived from a different bacteriophage (from
which is derived
the C-terminal region of the chimeric RBP) is also resistant to proteolytic
digestion as defined
above. Indeed, as explained above, it is shown that, even if these "wild-type"
RBPs are resistant
to proteolytic digestion, using isolated regions from these stable RBPs to
produce chimeras may
lead to the production of a chimera which is not resistant to proteolytic
digestion.
[63] By "N-terminal region" of a STF protein from a bacteriophage is meant
herein an amino
acid region of said STF protein starting at the N-terminal end of said STF
protein and ending at
positions 80-150, 320-460 or 495-560 of said STF protein, said positions being
with reference
to the lambda bacteriophage STF sequence (SEQ ID NO: 8). By "C-terminal
region" of a STF
protein from a bacteriophage is meant herein an amino acid region of said STF
protein starting
at positions 25-150, 320-460 or 495-560 of said STF protein, said positions
being with reference
to the lambda bacteriophage STF sequence (SEQ ID NO: 8), and ending at the C-
terminal end
of said STF protein.
[64] In a particular embodiment, the N-terminal region of a STF protein
derived from a
lambdoId bacteriophage corresponds to amino acids 1 to 528 of the lambda STF
protein of
sequence SEQ ID NO: 8.
[65] In a particular embodiment, the C-terminal region of said STF protein
derived from a
different bacteriophage corresponds to amino acids 218 to 875 of the STF
protein of sequence
SEQ ID NO: 16.
[66] In another particular embodiment, the C-terminal region of said STF
protein derived from
a different bacteriophage corresponds to amino acids 208 to 875 of the STF
protein of sequence
SEQ ID NO: 16.
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[67] In an alternative embodiment, the C-terminal region of said STF protein
derived from a
different bacteriophage corresponds to amino acids 28 to 632 of the STF
protein of sequence
SEQ ID NO: 12.
[68] In an alternative embodiment, the C-terminal region of said STF protein
derived from a
different bacteriophage corresponds to amino acids 62 to 632 of the STF
protein of sequence
SEQ ID NO: 12.
[69] In an embodiment, the chimeric STF protein comprises an N-terminal region
of a STF
protein derived from a lambdoId bacteriophage, preferably from a lambda or
lambda-like
bacteriophage, fused through said designed linker region to a C-terminal
region of a different
STF protein wherein said N-terminal region of the chimeric STF protein is
fused to said C-
terminal region of a different STF protein within one of the amino acids
regions selected from
positions 80-150, 320-460, or 495-560 of the N-terminal region with reference
to the lambda
bacteriophage STF sequence (SEQ ID NO: 8). In one aspect, the STF protein from
the lambdoId
bacteriophage, in particular from the lambda or lambda-like bacteriophage, and
the STF protein
derived from a different bacteriophage contain homology in one or more of
three amino acids
regions ranging from positions 80-150, 320-460, and 495-560 of the RBP with
reference to the
lambda bacteriophage STF sequence (SEQ ID NO: 8). In certain aspects, the
homology is
around 35% identity for 45 amino acids or more, around 50% identify for 30
amino acids or more,
or around 90% identity for 18 amino acids or more within the one or more of
three amino acids
regions ranging from positions 80-150, 320-460, and 495-560 of the STF protein
with reference
to the lambda bacteriophage STF sequence. In one specific aspect, the C-
terminal region of
the chimeric STF protein is derived from a bacteriophage or a bacteriocin. In
one aspect, the
chimeric STF protein comprises an N-terminal region of a STF protein fused to
a C-terminal
region of a STF protein derived from a different bacteriophage within one of
the amino acids
regions selected from positions 80-150, 320-460, or 495-560 of the N-terminal
STF region with
reference to the lambda bacteriophage STF sequence (SEQ ID NO: 8).
[70] In a particular embodiment, the chimeric RBP comprises an N-terminal
region of a STF
protein derived from a lambdoId bacteriophage, fused through a designed linker
region
consisting of 1 to 70 amino acids, more particularly of 1 to 30 amino acids,
to a C-terminal region
of a STF protein derived from a different bacteriophage, wherein said N-
terminal region and C-
terminal region are fused within a site of the N-terminal STF region, called
insertion site, having
at least 80%, 85%, 90%, 95%, 99% or 100% identity with a site selected from
the group
consisting of amino acids SAGDAS (SEQ ID NO: 1), ADAKKS (SEQ ID NO: 2), MDETNR
(SEQ
ID NO: 3), SASAAA (SEQ ID NO: 4), and GAGENS (SEQ ID NO: 5). In a particular
embodiment,
said insertion site has at least 80%, 85%, 90%, 95%, 99% or 100% identity with
the site of
sequence GAGENS (SEQ ID NO: 5).
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[71] In a particular embodiment, the chimeric RBP provided herein is an
engineered branched
receptor binding multi-subunit protein complex ("branched-RBP"). The
engineered chimeric
branched-RBP typically comprises two or more associated RBPs, derived from
bacteriophages,
which associate with one another based on the presence of interaction domains
(IDs). The
association of one subunit with another can be non-covalent or covalent. Each
of the polypeptide
subunits contain IDs that function as "anchors" for association of one subunit
RBP with another.
In specific embodiments, the chimeric branched-RBP may comprise multiple RBP
subunits,
including, for example, two, three, four, etc. subunits.
[72] The individual RBP subunit may bring different biological functions to
the overall
engineered chimeric branched-RBP. Such functions include but are not limited
to host
recognition and enzymatic activity. Such enzymatic activity includes
depolymerase activity. The
two or more associated receptor binding proteins of the chimeric branched-RBP
include, but are
not limited to, chimeric RBPs described herein that comprise a fusion between
the N-terminal
region of a RBP derived from a lambdoId bacteriophage, in particular from a
lambda or lambda-
like bacteriophage, and the C-terminal region of a RBP derived from a
different bacteriophage
wherein said chimeric RBP further comprises an ID domain.
[73] In an alternative embodiment, said chimeric RBP is a chimeric gpJ
protein.
Designed linker region
[74] By "designed linker region" is meant herein a region consisting of 1
to 70 amino acids,
more particularly 1 to 65 amino acids, still particularly 1 to 60 amino acids,
still particularly 1 to
55 amino acids, still particularly 1 to 50 amino acids, still particularly 1
to 45 amino acids, still
particularly 1 to 40 amino acids, still particularly 1 to 35 amino acids,
still particularly 1 to 30
amino acids, more particularly of 10 to 25 amino acids, or of 15 to 20 amino
acids, in particular
of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino
acids, which links the
N-terminal portion of the chimeric RBP and the C-terminal portion of the
chimeric RBP.
[75] In a particular embodiment, said designed linker region comprises the
insertion site as
defined above. In an alternative embodiment, said designed linker region is
adjacent to the
insertion site, as defined above. In a more particular embodiment, said
designed linker region is
at the C-terminal end of the insertion site as defined above. In other words,
in that embodiment,
the designed linker region starts at the amino acid directly following the
last amino acid of the
insertion site.
[76] In a particular embodiment, said designed linker region is part of the
N-terminal region
or of the C-terminal region of the chimeric RBP. In a particular aspect of
that embodiment, said
N-terminal region or said C-terminal region of the chimeric RBP comprises the
sequence of the
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linker region but said sequence has been specifically engineered (i.e.
modified), compared to
the corresponding wild-type sequence in the N-terminal region or C-terminal
region of the RBP
from which it is derived. Accordingly, in that particular aspect, when said
designed linker region
is part of the N-terminal region or of the C-terminal region of the chimeric
RBP, the sequence of
this designed linker region is not 100% identical to the sequence of the
corresponding region in
the N-terminal region of the RBP from which the N-terminal region of the
chimeric RBP is derived
or to the sequence of the corresponding region in the C-terminal region of the
RBP from which
the C-terminal region of the chimeric RBP is derived.
[77] In a particular embodiment, said linker region is engineered in such a
way as at least one
amino acid of the linker region which is likely to be targeted by trypsin
and/or chymotrypsin, as
defined above, is mutated.
[78] Accordingly, in a particular embodiment, at least one amino acid of the
designed linker
region, corresponding to an amino acid of the wildtype region sequence which
is likely to be
targeted by trypsin and/or chymotrypsin, is mutated compared to the wildtype
region sequence.
[79] In a particular embodiment said amino acid which is likely to be
targeted to trypsin and/or
chymotrypsin is selected from lysin (K), arginine (R), phenylalanine (F),
tryptophan (W), tyrosine
(Y) leucine (L) and methionine (M). In a particular embodiment, said amino
acid is substituted
by an alanine (A) or by any amino acid which is not lysin, arginine,
phenylalanine, tryptophan,
tyrosine, leucine or methionine, such as by an histidine (H).
[80] In a particular embodiment, only one amino acid of the designed linker
region is mutated.
In an alternative embodiment, more than one amino acid of the designed linker
region is mutated,
in particular at least two or at least three amino acids of the designed
linker region are mutated.
[81] In a particular embodiment, said linker region is part of the C-
terminal region of the
chimeric RBP and said at least one amino acid is located within the 15 first
amino acids of the
linker region. In that embodiment, said at least one amino acid is in
particular located within the
15 amino acids following the insertion site, as defined above.
[82] In a particular embodiment, said chimeric RBP, typically including
such designed linker
region, comprises or consists of the sequence SEQ ID NO: 9 (herein called STF-
V10-[FA]) or
SEQ ID NO: 10 (herein called STF-V10-[AAH]).
[83] In an alternative embodiment, said linker region is designed in such a
way as it comprises
a structure which is resistant to proteolytic digestion, and which thus
typically restores the
proteolytic digestion resistance of the chimeric RBP compared to a chimeric
RBP which differs
only by the absence of said linker region.
[84] Therefore, In a particular aspect of the embodiment wherein said designed
linker region
is part of the N-terminal region or of the C-terminal region of the chimeric
RBP, said N-terminal
region or said C-terminal region of the chimeric RBP comprises the sequence of
the linker region,
preferably respectively at their C-terminal part or N-terminal part, said
sequence being identical
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to the corresponding sequence in the N-terminal region or C-terminal region of
the RBP from
which it is derived, and said sequence restoring resistance to proteolytic
digestion, as defined
above, to said chimeric RBP compared to a chimeric RBP only differing by the
absence of said
linker region.
[85] In other words, in that particular aspect, said designed linker region
is part of the N-
terminal region or of the C-terminal region of the chimeric RBP, and the
sequence of this
designed linker region has not been modified compared to the wild-type
sequence in the N-
terminal region or C-terminal region of the RBP from which it is derived, but
has been specifically
selected to be present, preferably at the C-terminal part of the N-terminal
region or at the N-
terminal part of the C-terminal region, compared to an N-terminal region or a
C-terminal region
not including it, because of its resistance to proteolytic digestion as
defined above.
[86] Alternatively, in a particular embodiment, said designed linker region
comprises or
consists of an heterologous amino acid sequence which is not derived from one
of the RBP from
which the N-terminal region and the C-terminal region of the chimeric RBP are
derived. In a
particular embodiment, said designed linker region comprises or consists of a
sequence which
is derived from a RBP which is not one of the RBP from which the N-terminal
region and the C-
terminal region of the chimeric RBP are derived.
[87] In a particular embodiment, said designed linker region consists of 10
to 70 amino acids,
in particular of 10 to 65 amino acids, of 10 to 64 amino acids, of 10 to 63
amino acids, of 10 to
62 amino acids, of 10 to 61 amino acids, of 10 to 60 amino acids, of 10 to 55
amino acids, of 10
to 50 amino acids, of 10 to 45 amino acids, of 10 to 40 amino acids, of 10 to
35 amino acids, of
10 to 30 amino acids, of 10 to 20 amino acids, in particular of 11 to 20 amino
acids, or of 12 to
amino acids.
[88] In a particular embodiment, said designed linker region comprises or
consists of an
amino acid sequence GSATDVMIQL (SEQ ID NO: 6) or GSATDVMIQLA (SEQ ID NO: 7),
herein
called helix sequence.
[89] In a particular embodiment, said sequence is located within the 10 or
12 first amino acids
of the designed linker region. In a more particular embodiment, said sequence
is located directly
after the insertion site, as defined above.
[90] In a particular embodiment, said chimeric RBP, typically including
such designed linker
region, comprises or consists of the sequence SEQ ID NO: 11 (herein called STF-
V10-[Helix]).
In another embodiment, said chimeric RBP, typically including such designed
linker region,
comprises or consists of the sequence SEQ ID NO: 13 (herein called K5 5.0) or
SEQ ID NO: 14
(herein called K5 5.1).
[91] In a particular embodiment, said designed linker region comprises or
consists of the
amino acid sequence SEQ ID NO: 34 or SEQ ID NO: 36. In a particular
embodiment, said
sequence is located directly after the insertion site, as defined above. In a
particular
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embodiment, said chimeric RBP, typically including such designed linker
region, comprises or
consists of the sequence SEQ ID NO: 38 (herein called K5 9.0) or SEQ ID NO: 40
(herein called
K5 9.1).
[92] In a particular embodiment, the designed linker region comprises a
helix or helical
bundle.
[93] By "helical bundle" or "helix bundle" is meant herein a small protein
fold composed of
several alpha helices that are usually nearly parallel or antiparallel to each
other.
[94] By "helix" is meant herein a motif in the secondary structure of
proteins.
[95] The present disclosure also provides a nucleic acid encoding a chimeric
RBP as defined
above.
[96] In a particular embodiment, said nucleic acid encodes a chimeric RBP
comprising or
consisting of the sequence SEQ ID NO: 9 and typically comprises or consists of
the sequence
SEQ ID NO: 17. In another particular embodiment, said nucleic acid encodes a
chimeric RBP
comprising or consisting of the sequence SEQ ID NO: 10 and typically comprises
or consists of
the sequence SEQ ID NO: 18. In another particular embodiment, said nucleic
acid encodes a
chimeric RBP comprising or consisting of the sequence SEQ ID NO: 11 and
typically comprises
or consists of the sequence SEQ ID NO: 19. In another particular embodiment,
said nucleic acid
encodes a chimeric RBP comprising or consisting of the sequence SEQ ID NO: 13
and typically
comprises or consists of the sequence SEQ ID NO: 20. In another particular
embodiment, said
nucleic acid encodes a chimeric RBP comprising or consisting of the sequence
SEQ ID NO: 14
and typically comprises or consists of the sequence SEQ ID NO: 21. In another
particular
embodiment, said nucleic acid encodes a chimeric RBP comprising or consisting
of the
sequence SEQ ID NO: 38 and typically comprises or consists of the sequence SEQ
ID NO: 39.
In another particular embodiment, said nucleic acid encodes a chimeric RBP
comprising or
consisting of the sequence SEQ ID NO: 40 and typically comprises or consists
of the sequence
SEQ ID NO: 41.
[97] Such nucleic acids may be included in vectors such as bacteriophages,
plasmids,
phagemids, phage-plasmids, viruses, and other vehicles which enable transfer
and expression
of the chimeric RBP encoding nucleic acids. The present disclosure thus also
provides such a
vector comprising a nucleic acid encoding a chimeric RBP as defined above, in
particular
comprising a nucleic acid encoding a chimeric RBP comprising or consisting of
the sequence
SEQ ID NO: 11 which typically comprises or consists of the sequence SEQ ID NO:
19.
LambdaId bacterial delivery vehicle
[98] The present disclosure relates to a lambdoId bacterial delivery vehicle,
typically for use
in in vivo delivery of a DNA payload of interest into a targeted bacterial
cell, wherein said
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lambdoId delivery vehicle comprises a chimeric RBP resistant to proteolytic
digestion, in
particular within the gastrointestinal tract, as defined in the section
"Chimeric RBP' above.
[99] The bacterial delivery vehicles provided herein enable transfer of a
nucleic acid payload,
encoding a protein or nucleic acid of interest, into a desired target
bacterial host cell.
Delivery vehicle
[100] As used herein, the term "delivery vehicle" refers to any means that
allows the transfer
of a payload into a bacterium. There are several types of delivery vehicles
encompassed by the
present disclosure including, without limitation, bacteriophage scaffold,
virus scaffold, chemical
based delivery vehicle (e.g., cyclodextrin, calcium phosphate, cationic
polymers, cationic
liposomes), protein-based or peptide-based delivery vehicle, lipid-based
delivery vehicle,
nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles
(e.g.,
transformation, electroporation, sonoporation, optical transfection), particle-
based delivery
vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment,
cell-penetrating
peptides) or donor bacteria (conjugation). Any combination of delivery
vehicles is also
encompassed by the present disclosure. The delivery vehicle can refer to a
bacteriophage
derived scaffold and can be obtained from a natural, evolved or engineered
capsid.
[101] The bacterial delivery vehicles provided herein which enable transfer of
a nucleic acid
payload, encoding a protein or nucleic acid of interest, into a desired target
bacterial host cell
are characterized by having a chimeric RBP resistant to proteolytic digestion,
in particular within
the gastrointestinal tract, as defined in the section "Chimeric RBP' above.
[102] In a particular embodiment, said chimeric RBP is a chimeric STF protein
as defined in
the section "Chimeric RBP' above. In a particular embodiment, said chimeric
STF protein is a
functional STF protein.
[103] As used herein, a functional protein means in general a protein with a
biological activity;
more specifically a functional chimeric protein relates to a chimeric protein
contributing to the
efficient delivery of a DNA payload into a target strain. The efficiency
threshold depends on a
number of factors such as the type of protein, type of target strain and type
of environment. For
instance, STF and gpJ proteins allow for recognition, binding (and in some
cases also
degradation) of an extracellular epitope such as LPS, capsules and outer
membrane proteins;
gpH proteins allow for an efficient injection and hence successful passage of
the DNA payload
through the periplasm.
[104] In some embodiments, the bacterial delivery vehicles disclosed herein
further comprise
the corresponding natural chaperone proteins (designated "accessory proteins"
or "AP") of the
chimeric RBPs. Such AP proteins assist in the folding of the chimeric RBPs.
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[105] In a particular embodiment, the chimeric STF protein has enzyme activity
such as
depolymerase activity and the bacterial cell population of interest comprises
encapsulated
bacteria.
[106] Bacterial delivery vehicles are also provided that further comprise
recombinant gpJ
proteins. Such gpJ proteins include recombinant gpJ proteins, including
chimeric proteins as
defined in the section "Chimeric RBP' above, that permit recognition of a
bacterial cell receptor
other than the LamB OMP receptor. It is known that receptor-recognition
activity of gpJ lies in
the C-terminal part of the protein, with a fragment as small as 249 amino
acids conferring
capability of binding to the LamB receptor [10]. In a particular embodiment,
such chimeric gpJ
protein may comprise a fusion between the N-terminal region of a gpJ protein
from a lambdoId
bacteriophage, in particular from a lambda or lambda-like bacteriophage, and
the C-terminal
region of a different gpJ protein.
[107] By "N-terminal region" of a gpJ protein from a bacteriophage is meant
herein an amino
acid region of said gpJ protein starting at the N-terminal end of said gpJ
protein and ending at
positions 810-825 or 950-970 of said gpJ protein, said positions being with
reference to the
lambda bacteriophage gpJ protein sequence (SEQ ID NO: 22). By "C-terminal
region" of a gpJ
protein from a bacteriophage is meant herein an amino acid region of said gpJ
protein starting
at positions 810-825 or 950-970 of said gpJ protein, said positions being with
reference to the
lambda bacteriophage gpJ protein sequence (SEQ ID NO: 22), and ending at the C-
terminal end
of said gpJ protein.
[108] For production of chimeric gpJ proteins, two insertion points, located
respectively at
positions corresponding to amino acids 814-821 and 958-966 of the lambda
bacteriophage gpJ
protein sequence (SEQ ID NO: 22) have previously been identified by the
inventors. In non-
limiting aspects, such insertion sites may be utilized for production of
chimeric proteins. Both
insertion points yield functional gpJ chimeras with altered receptor binding.
In an embodiment,
the bacterial delivery vehicles contain a chimeric gpJ protein comprising a
fusion between an N-
terminal region of a gpJ protein derived from a lambdoId bacteriophage, in
particular from a
lambda or lambda-like bacteriophage, and a C-terminal region of a different
gpJ protein wherein
said N-terminal region of the chimeric gpJ protein is fused to said C-terminal
region of a different
gpJ protein within one of the amino acids regions selected from positions 810-
825, or 950-970
of the N-terminal region with reference to the lambda bacteriophage gpJ
protein sequence (SEQ
ID NO: 22).
[109] In a specific embodiment, the chimeric gpJ protein comprises a fusion
between the N-
terminal region of a lambda bacteriophage gpJ protein and the C-terminal
region of a gpJ protein
from a different bacteriophage, which typically recognizes and binds OmpC,
said N-terminal
region being in particular fused to said C-terminal region within the amino
acid region 950-970
of the N-terminal region with reference to the lambda bacteriophage gpJ
protein sequence (SEQ
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ID NO: 22). In said embodiment, the chimeric gpJ variant may be H591
comprising or consisting
of the amino acid sequence SEQ ID NO: 23 and typically encoded by the
nucleotide sequence
SEQ ID NO: 24, said H591 chimeric gpJ variant typically recognizing and
binding OmpC. In
another embodiment, the chimeric gpJ protein comprises a fusion between the N-
terminal region
of a lambda bacteriophage gpJ protein and the C-terminal region of a gpJ
protein from a different
bacteriophage, which typically recognizes a receptor present in 0157 strains,
said N-terminal
region being in particular fused to said C-terminal region within the amino
acid region 810-825
of the N-terminal region with reference to the lambda bacteriophage gpJ
protein sequence (SEQ
ID NO: 22). In said embodiment, the chimeric gpJ variant may be Z2145
comprising or consisting
of the amino acid sequence SEQ ID NO: 25 and typically encoded by the
nucleotide sequence
SEQ ID NO: 26, said Z2145 chimeric gpJ variant typically recognizing a
receptor present in 0157
strains. In still another embodiment, the chimeric gpJ protein comprises a
fusion between the N-
terminal region of a lambda bacteriophage gpJ protein and the C-terminal
region of a gpJ protein
from a different bacteriophage, which typically recognizes the OmpC receptor
present in 0157
strains, said N-terminal region being in particular fused to said C-terminal
region within the amino
acid region 950-970 of the N-terminal region with reference to the lambda
bacteriophage gpJ
protein sequence (SEQ ID NO: 22). In said embodiment, the chimeric gpJ variant
may be the
"1A2" variant comprising or consisting of the of amino acid sequence SEQ ID
NO: 27 and
typically encoded by the nucleotide sequence SEQ ID NO: 28, said 1A2 chimeric
gpJ variant
typically recognizing the OmpC receptor present in 0157 strains. In still
another embodiment,
the chimeric gpJ protein comprises a fusion between the N-terminal region of a
lambda
bacteriophage gpJ protein and the C-terminal region of a gpJ protein from a
different
bacteriophage, which typically recognizes the OmpC receptor present in both
0157 and
MG1655 strains, said N-terminal region being in particular fused to said C-
terminal region within
the amino acid region 950-970 of the N-terminal region with reference to the
lambda
bacteriophage gpJ protein sequence (SEQ ID NO: 22). In said embodiment, the
chimeric gpJ
variant may be the "A8" variant comprising or consisting of the amino acid
sequence SEQ ID
NO: 29 and typically encoded by the nucleotide sequence SEQ ID NO: 30, said A8
chimeric gpJ
variant typically recognizing the OmpC receptor in both 0157 and MG1655
strains.
[110] Bacterial delivery vehicles are also provided that further comprise
recombinant gpH
proteins. Such gpH proteins include recombinant gpH proteins that permit or
allow improved
entry of bacterial vectors in cells having deficiencies or alterations in
permease complexes. One
such variant is the "gpH-IAI" variant of amino acid sequence SEQ ID NO: 31.
[111] In a particular embodiment, said bacterial delivery vehicle comprises
chimeric STF of
sequence SEQ ID NO: 11 and chimeric gpJ variant of sequence SEQ ID NO: 27.
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[112] In a particular embodiment, the lambdoId delivery vehicle as disclosed
above further
comprises a functional lambdoId bacteriophage gpJ protein, as defined above,
and/or a
functional lambdoId bacteriophage gpH protein, as defined above.
[113] In aspects, the bacterial delivery vehicles provided herein, are
vehicles wherein the one
or more of the chimeric STF protein, the gpJ protein and/or the gpH protein
are further
engineered to increase the efficiency of transfer of the DNA payload into the
targeted bacterial
cell population. Such bacterial cell populations include for example E. coll,
and other bacterial
species of interest.
[114] In a particular embodiment, the delivery vehicle is incapable of self-
reproduction.
[115] In the context of the present invention, "self-reproduction" is
different from "self-
replication", "self-replication" referring to the capability of replicating a
nucleic acid, whereas
"self-reproduction" refers to the capability of having a progeny, in
particular of producing new
delivery vehicles, said delivery vehicles being either produced empty or with
a nucleic acid of
interest packaged.
[116] By "delivery vehicle incapable of self-reproduction" is meant herein
that at least one,
several or all functional gene(s) necessary to produce said delivery vehicle
is(are) absent from
said delivery vehicle (and from said vector included in said delivery
vehicle). In a preferred
embodiment, said at least one, several or all functional gene(s) necessary to
produce said
delivery vehicle is(are) present in the donor cell as defined above,
preferably in a plasmid, in the
chromosome or in a helper phage present in the donor cell as defined below,
enabling the
production of said delivery vehicle in said donor cell.
[117] In the context of the invention, said functional gene(s) necessary to
produce said delivery
vehicle may be absent through (i) the absence of the corresponding gene or
(ii) the presence of
the corresponding gene but in a non-functional form.
[118] In an embodiment, the sequence of said gene necessary to produce said
delivery vehicle
is absent from said delivery vehicle. In a preferred embodiment, the sequence
of said gene
necessary to produce said delivery vehicle has been replaced by a nucleic acid
sequence of
interest.
[119] Alternatively, said gene necessary to produce said delivery vehicle is
present in said
delivery vehicle in a non-functional form, for example in a mutant non-
functional form, or in a
non-expressible form, for example with deleted or mutated non-functional
regulators. In a
preferred embodiment, said gene necessary to produce said delivery vehicle is
present in said
delivery vehicle in a mutated form which renders it non-functional in the
target cell, while
remaining functional in the donor cell.
[120] In the context of the invention, genes necessary to produce said
delivery vehicle
encompass any coding or non-coding nucleic acid required for the production of
said delivery
vehicle.
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[121] Examples of genes necessary to produce said delivery vehicle include
genes encoding
phage structural proteins; phage genes involved in the control of genetic
expression; phage
genes involved in transcription and/or translation regulation; phage genes
involved in phage
DNA replication; phage genes involved in production of phage proteins; phage
genes involved
in phage proteins folding; phage genes involved in phage DNA packaging; and
phage genes
encoding proteins involved in bacterial cell lysis.
Packaged phagemids
[122] Delivery vehicles include packaged phagemids, as well as bacteriophage,
as disclosed
herein. An eligobiotic is a packaged phagemid, i.e a payload encapsidated in
a phage-derived
capsid. The engineering of such delivery vehicles is well known to those
skilled in the art. Such
engineering techniques may employ production cell lines engineered to express
the STF, gpJ
and gpH proteins disclosed herein. The present disclosure thus also provides a
production cell
line expressing the chimeric RBPs provided herein.
[123] In one aspect, bacterial delivery vehicles with desired target host
ranges are provided for
use in transfer of a payload to the microbiome of a host. The bacterial
delivery vehicles may be
characterized by combinations of chimeric STF, and wild-type and engineered
gpJ and gpH
proteins.
[124] Generation of packaged phagemids and bacteriophage particles are routine
techniques
well-known to one skilled in the art. In an embodiment, a satellite phage
and/or helper phage
may be used to promote the packaging of the payload in the delivery vehicles
disclosed herein.
Helper phages provide functions in trans and are well known to the man skilled
in the art. The
helper phage comprises all the genes coding for the structural and functional
proteins that are
indispensable for the payload to be packaged, (i.e. the helper phage provides
all the necessary
gene products for the assembly of the delivery vehicle). The helper phage may
contain a
defective origin of replication or packaging signal, or completely lack the
latter, and hence it is
incapable of self-packaging, thus only bacterial delivery particles carrying
the payload or plasmid
will be produced. Helper phages may be chosen so that they cannot induce lysis
of the host
used for the delivery particle production. One skilled in the art would
understand that some
bacteriophages are defective and need a helper phage for payload packaging.
Thus, depending
on the bacteriophage chosen to prepare the bacterial delivery particles, the
person skilled in the
art would know if a helper phage is required. Sequences coding for one or more
proteins or
regulatory processes necessary for the assembly or production of packaged
payloads may be
supplied in trans. For example, the STF, gpJ and gpH proteins of the present
disclosure may be
provided in a plasmid under the control of an inducible promoter or expressed
constitutively. In
this case, the phage wild-type sequence may or not contain a deletion of the
gene or sequence
supplied in trans. Additionally, chimeric or modified phage sequences encoding
a new function,
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like an engineered STF, gpJ or gpH protein, may be directly inserted into the
desired position in
the genome of the helper phage, hence bypassing the necessity of providing the
modified
sequence in trans. Methods for both supplying a sequence or protein in trans
in the form of a
plasmid, as well as methods to generate direct genomic insertions,
modifications and mutations
are well known to those skilled in the art.
[125] In a particular embodiment, said helper phage comprises a nucleic acid
sequence
encoding the chimeric RBP comprising or consisting of the sequence SEQ ID NO:
11, said
nucleic acid sequence typically comprising or consisting of the sequence SEQ
ID NO: 19, and
said helper phage optionally further comprises a nucleic acid sequence
encoding the chimeric
gpJ variant comprising or consisting of the sequence SEQ ID NO: 27, said
nucleic acid sequence
typically comprising or consisting of the sequence SEQ ID NO: 28.
[126] In a particular embodiment, said helper phage is a lambda prophage
wherein (i) the
nucleic acid encoding a wild-type STF protein has been replaced by a nucleic
acid sequence
encoding the chimeric RBP comprising or consisting of the sequence SEQ ID NO:
11, said
nucleic acid sequence typically comprising or consisting of the sequence SEQ
ID NO: 19, (ii) the
nucleic acid encoding a wild-type gpJ protein has been replaced by a nucleic
acid sequence
encoding the chimeric gpJ variant comprising or consisting of the sequence SEQ
ID NO: 27,
said nucleic acid sequence typically comprising or consisting of the sequence
SEQ ID NO: 28,
and (iii) the Cos site has been removed, and wherein optionally (iv) the
helper prophage contains
a mutation which prevents spontaneous cell lysis, such as the 5am7 mutation
and (v) the helper
prophage contains a thermosensitive version of the master cl repressor, such
as the c1857
version.
[127] Another object of the disclosure thus also concerns providing a
production cell line, as
defined above, comprising a helper phage as defined above.
[128] In a particular embodiment, said bacterial delivery vehicle comprises
said DNA payload
of interest.
Payload
[129] As used herein, the term "payload" refers to any nucleic acid sequence
or amino acid
sequence, or a combination of both (such as, without limitation, peptide
nucleic acid or peptide-
oligonucleotide conjugate) transferred into a bacterium with a delivery
vehicle. The term
"payload" may also refer to a plasmid, a vector or a cargo. The payload can be
a phagemid or
phasmid obtained from natural, evolved or engineered bacteriophage genome. The
payload can
also be composed only in part of phagemid or phasmid obtained from natural,
evolved or
engineered bacteriophage genome.
[130] In a particular embodiment, the payload has a size superior or equal to
4 kbp, and
preferably inferior or equal to 51 kb.
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[131] In said embodiment, the payload may have a size, an integer multiple of
which is between
36 kb and 51 kb. In other words, in that embodiment, there is at least an
integer n, such as
36 kb n x size of the payload 51 kb .
[132] As described herein it is more particularly demonstrated that it is
possible to produce a
more uniform population of bacterial delivery vehicles comprising an almost
unique number of
payload copies when said payload has a size of a specific range.
[133] In a particular embodiment, the payload has a size strictly superior to
10.000 kb and
strictly inferior to 12.000 kb. In an alternative embodiment, the payload has
a size strictly superior
to 12.500 kb and strictly inferior to 16.667 kb, in particular a size strictly
superior to 12.500 kb
and inferior to 13.000 kb.
[134] In another particular embodiment, the payload has a size superior or
equal to 18.000 kb
and inferior or equal to 25.000 kb, in particular inferior or equal to 24.000
kb.
[135] In a particular embodiment, said payload has a size of 11.6 kb.
[136] The payload may be a nucleic acid plasmid that is able to circularize
upon transfer into
the target cell and then either replicate or integrate inside the chromosome.
Replication of the
vector DNA is dependent on the presence of a bacterial origin of replication.
Once replicated,
inheritance of the plasmid into each of the daughter cells can be mediated by
the presence of
an active partitioning mechanism and a plasmid addiction system such as
toxin/anti-toxin
system.
[137] As used herein, the term "nucleic acid" refers to a sequence of at least
two nucleotides
covalently linked together which can be single-stranded or double-stranded or
contains
portion(s) of both single-stranded and double-stranded sequence. Nucleic acids
can be naturally
occurring, recombinant or synthetic. The nucleic acid can be in the form of a
circular sequence
or a linear sequence or a combination of both forms. The nucleic acid can be
DNA, both genomic
or cDNA, or RNA or a combination of both. The nucleic acid may contain any
combination of
deoxyribonucleotides and ribonucleotides, and any combination of bases,
including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine,
isocytosine, 5-
hydroxymethylcytosine and isoguanine. Other examples of modified bases that
can be used are
detailed in Chemical Reviews 2016, 116 (20) 12655-12687. The term "nucleic
acid" also
encompasses any nucleic acid analogs which may contain other backbones
comprising, without
limitation, phosphoramide, phosphorothioate, phosphorodithioate, 0-
methylphosphoroamidite
linkage and/or deoxyribonucleotides and ribonucleotides nucleic acids. Any
combination of the
above features of a nucleic acid is also encompassed by the present
disclosure.
[138] Origins of replication known in the art have been identified from
species-specific plasmid
DNAs (e.g. ColE1, RI, pT181, pSC101, pMB1, R6K, RK2, p15a and the like), from
bacterial virus
(e.g. TX174, M13, F1 and P4) and from bacterial chromosomal origins of
replication (e.g. oriC).
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In one embodiment, the phagemid according to the disclosure comprises a
bacterial origin of
replication that is functional in the targeted bacteria.
[139] Alternatively, the plasmid according to the disclosure does not comprise
any functional
bacterial origin of replication or contain an origin of replication that is
inactive in the targeted
bacteria. Thus, the plasmid of the disclosure cannot replicate by itself once
it has been
introduced into a bacterium by the bacterial virus particle.
[140] In one embodiment, the origin of replication on the plasmid to be
packaged is inactive in
the targeted bacteria, meaning that this origin of replication is not
functional in the bacteria
targeted by the bacterial virus particles, thus preventing unwanted plasmid
replication.
[141] In one embodiment, the plasmid comprises a bacterial origin of
replication that is
functional in the bacteria used for the production of the bacterial virus
particles.
[142] Plasmid replication depends on host enzymes and on plasmid-controlled
cis and trans
determinants. For example, some plasmids have determinants that are recognized
in almost all
gram-negative bacteria and act correctly in each host during replication
initiation and regulation.
Other plasmids possess this ability only in some bacteria (Kues, U and Stahl,
U 1989 Microbiol
Rev 53:491-516).
[143] Plasmids are replicated by three general mechanisms, namely theta type,
strand
displacement, and rolling circle (reviewed by Del Solar et al. 1998 Microhio
and Molec Biol. Rev
62:434-464) that start at the origin of replication. These replication origins
contain sites that are
required for interactions of plasmid and/or host encoded proteins.
[144] Origins of replication used on the plasmid of the disclosure may be of
moderate copy
number, such as colElori from pBR322 (15-20 copies per cell) or the R6K
plasmid (15-20 copies
per cell) or may be high copy number, e.g. pUC oris (500-700 copies per cell),
pGEM oris (300-
400 copies per cell), pTZ oris (>1000 copies per cell) or pBluescript oris
(300-500 copies per
cell).
[145] In one embodiment, the bacterial origin of replication is selected in
the group consisting
of ColE1, pMB1 and variants (pBR322, pET, pUC, etc), p15a, ColA, ColE2, pOSAK,
pSC101,
R6K, IncW (pSa etc), IncF1I, pT181, P1, F IncP, IncC, IncJ, IncN, IncP1,
IncP4, IncQ, IncH11,
RSF1010, CloDF13, NTP16, R1, f5, pPS10, p0194, pE194, BBR1, pBC1, pEP2, pWV01,
pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22, pAMbeta1,
pIP501,
pIP407, ZM6100(Sa), pCU1, RA3, pM0L98, RK2/RP4/RP1/R68, pB10, R300B, pR01614,
pR01600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl,
RA1,
TF-FC2, pMV158 and pUB113.
[146] In an embodiment, the bacterial origin of replication is a E.coli origin
of replication
selected in the group consisting of ColE1, pMB1 and variants (pBR322, pET,
pUC, etc), p15a,
ColA, ColE2, pOSAK, pSC101, R6K, IncW (pSa etc), IncF1I, pT181, P1, F IncP,
IncC, IncJ, IncN,
IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5 and pPS10.
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[147] In an embodiment, the bacterial origin of replication is selected in the
group consisting of
p0194, pE194, BBR1, pBC1, pEP2, pWV01, pLF1311, pAP1, pWKS1, pLS1, pLS11,
pUB6060,
pJD4, pIJ101, pSN22, pAMbetal , pIP501, pIP407, ZM6100(Sa), pCUl , RA3,
pM0L98,
RK2/RP4/RP1/R68, pB10, R300B, pR01614, pR01600, pECB2, pCM1, pFA3, RepFIA,
RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.
[148] In an embodiment, the bacterial origin of replication is ColEl.
[149] The delivered nucleic acid sequence according to the disclosure may
comprise a phage
replication origin which can initiate, with complementation of a complete
phage genome, the
replication of the delivered nucleic acid sequence for later encapsulation
into the different
capsids.
[150] A phage origin of replication comprised in the delivered nucleic acid
sequence of the
disclosure can be any origin of replication found in a phage.
[151] In an embodiment, the phage origin of replication can be the wild-type
or non-wildtype
sequence of the M13, f1, TX174, P4, lambda, P2, lambda-like, HK022, mEP237,
HK97, HK629,
HK630, mEP043, mEP213, mEP234, mEP390, mEP460, mEPx1 , mEPx2, phi80, mEP234,
T2,
T4, T5, T7, RB49, phiX174, R17, PRD1 P1-like, P2-like, P22, P22-like, N15 and
N15-like
bacteriophages.
[152] In an embodiment, the phage origin of replication is selected in the
group consisting of
phage origins of replication of M13, f1, TX174, P4, and lambda.
[153] In a particular embodiment, the phage origin of replication is the
lambda or P4 origin of
replication. In a particular embodiment, the phage origin of replication is
from Propionibacterium
phages: BW-like phages such as Doucette, B22, E6, G4, BV-like phages such as
Anatole, El,
B3, BX-like phages such as PFR1 and PFR2, filamentous B5 phage, BU-like phages
(Cutibacterium acnes phages).
[154] In a particular embodiment, the payload or vector comprises a
conditional origin of
replication which is inactive in the targeted bacteria but is active in a
donor bacterial cell.
[155] In the context of the invention, a "conditional origin of replication"
refers to an origin of
replication whose functionality may be controlled by the presence of a
specific molecule.
[156] In a particular embodiment, the conditional origin of replication is an
origin of replication,
the replication of which depends upon the presence of one or more given
protein, peptid, RNA,
nucleic acid, molecule or any combination thereof.
[157] In a particular embodiment, the replication of said origin of
replication may further depend
on a process, such as transcription, to activate said replication.
[158] In the context of the invention, said conditional origin of replication
is inactive in the
targeted bacteria because of the absence of said given protein, peptid, RNA,
nucleic acid,
molecule or any combination thereof in said targeted bacteria.
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[159] In a particular embodiment, said conditional origin of replication is
active in said donor
bacterial cell because said donor bacterial cell expresses said given protein,
peptid, RNA,
nucleic acid, molecule or any combination thereof. In a particular embodiment,
said protein,
peptid, RNA nucleic acid, molecule or any combination thereof is expressed in
trans in said
donor bacterial cell.
[160] By "in trans" is meant herein that said protein, peptid, RNA, nucleic
acid, molecule or any
combination thereof is not encoded on the same nucleic acid molecule as the
one comprising
the origin of replication. In a particular embodiment, said protein, peptid,
RNA, nucleic acid,
molecule or any combination thereof is encoded on a chromosome or on a vector,
in particular
a plasmid. In a particular embodiment, said vector comprises an antibiotic
resistance marker. In
an alternative embodiment, said vector is devoid of antibiotic resistance
marker.
[161] Since said conditional origin of replication is inactive in the targeted
bacteria because of
the absence of said given protein, peptid, RNA, nucleic acid, molecule or any
combination
thereof in said targeted bacteria, said conditional origin of replication may
be selected depending
on the specific bacteria to be targeted.
[162] The conditional origin of replication disclosed herein may originate
from plasmids,
bacteriophages or PICIs which preferably share the following characteristics:
they contain in
their origin of replication repeat sequences, or iterons, and they code for at
least one protein
interacting with said origin of replication (i.e. Rep, protein 0, protein P,
pri) which is specific to
them.
[163] By way of example, mention may be made of the conditional replication
systems of the
following plasmids and bacteriophages: RK2, R1 , pSC101, F, Rts1, RSF1010, P1,
P4, lambda,
phi82, phi80.
[164] In a particular embodiment, said conditional origin of replication is
selected from the
group consisting of the R6KA DNA replication origin and derivatives thereof,
the IncPa oriV origin
of replication and derivatives thereof, ColE1 origins of replication modified
to be under an
inducible promoter, and origins of replication from phage-inducible
chromosomal islands (PICIs)
and derivatives thereof.
[165] In a particular embodiment, said conditional origin of replication is an
origin of replication
present in less than 50%, or less than 40%, less than 30%, less than 20%, less
than 10% or less
than 5% of the bacteria of the host microbiome.
[166] In another particular embodiment, said conditional origin of replication
comprises or
consists of a sequence less than 80% identical, in particular less than 70%,
less than 60%, less
than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less
than 5% or less
than 1% identical to the sequences of the origins of replication of the
bacteria of the host
microbiome, in particular of the bacteria representing more than 50%, more
particularly more
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than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the
host
microbiome.
[167] As used herein, the term "phage-inducible chromosomal islands" or
"PICIs" refers to
mobile genetic elements having a conserved gene organization, and encode a
pair of divergent
regulatory genes, including a PIC1 master repressor. Typically, in Gram-
positive bacteria, left of
rpr, and transcribed in the same direction, PICIs encode a small set of genes
including an
integrase (int) gene; right of rpr, and transcribed in the opposite direction,
the PICIs encode an
excision function (xis), and a replication module consisting of a primase
homolog (pri) and
optionally a replication initiator (rep), which are sometimes fused, followed
by a replication origin
(on), next to these genes, and also transcribed in the same direction, PICIs
encode genes
involved in phage interference, and optionally, a terminase small subunit
homolog (terS).
[168] In a particular embodiment, said conditional origin of replication is an
origin of replication
derived from phage-inducible chromosomal islands (PICIs).
[169] A particular conditional origin of replication has indeed been derived
from PICIs.
[170] It was shown that it is possible to derive novel conditionally
replicative vectors or
payloads, in particular based on the primase-helicase and origin of
replication from PICIs. These
origins may be relatively rare in target strains, and more advantageously the
primase-ori pair
may be unique for each PIC!, significantly reducing the possibility of
undesired recombination or
payload spread events. They can further be modified to further limit
recombination chances and
remove restriction sites to bypass target bacteria defense systems.
[171] In a particular embodiment, said conditional origin of replication is
derived from the origin
of replication from the PIC1 of the Escherichia coli strain 0FT073, disclosed
in Fillol-Salom et al.
(2018) The ISME Journal 12:2114-2128.
[172] In a particular embodiment, said conditional origin of replication is
the primase on from
the PIC1 of the Escherichia coli strain 0FT073, typically of sequence SEQ ID
NO: 46.
[173] In another particular embodiment, said conditional origin of replication
is the primase on
from the PIC1 of the Escherichia coli strain 0FT073, devoid of at least 1, at
least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12,
at least 13, at least 14, at least 15 or at least 16 restriction site(s)
selected from the group
consisting of GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNTGG (SEQ ID NO:
47), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD,
GGCYAC, RGCCGGYYD, and VGCCGGYBD.
[174] In a particular embodiment, said conditional origin of replication is
the primase on from
the PIC1 of the Escherichia coli strain 0FT073, devoid of the restriction site
GAAABCC.
[175] Preferably, said conditional origin of replication is of sequence SEQ ID
NO: 48.
[176] In another particular embodiment, said conditional origin of replication
is the primase on
from the PIC1 of the Escherichia coli strain 0FT073 devoid of the restriction
sites GAAABCC,
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GCCGGC, RCCGGY, GCNGC, TWCANNNNNNTGG (SEQ ID NO: 47), TGGCCA, ACCYAC,
YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC,
RGCCGGYYD, and VGCCGGYBD. Preferably, said conditional origin of replication
is of
sequence SEQ ID NO: 49.
[177] In a particular embodiment, wherein said origin of replication is
derived from phage-
inducible chromosomal islands (PICIs), said conditional origin of replication
is active in said
donor bacterial cell because said donor bacterial cell expresses a rep
protein, in particular a
primase-helicase, in particular a primase-helicase of sequence SEQ ID NO: 50,
typically
encoded by a nucleic acid comprising or consisting of the sequence SEQ ID NO:
51.
[178] It was demonstrated that these specific conditional origins of
replication were particularly
compatible with lambda-based packaging, leading to sufficiently high titers
(>1010 /mL) required
for microbiota-related applications.
[179] In a particular embodiment, when said payload or vector is a phagemid,
said origin of
replication may be derived from a microorganism which is different from the
one that is used to
encode the structural elements of the capsid packaging said phagemid.
[180] By "donor bacterial cell" is meant herein a bacterium that is capable of
hosting a payload
or vector as defined above, of producing a payload or vector as defined above
and/or which is
capable of transferring said payload or vector as defined above to another
bacterium. In a
particular embodiment, said payload or vector may be a phagemid, and said
donor bacterial cell
may then be a bacterial cell able to produce said phagemid, more particularly
in the form of a
packaged phagemid.
[181] Preferably, said donor bacterial cell stably comprises said payload or
vector and is able
to replicate said payload or vector.
[182] In a particular embodiment, when the conditional origin of replication
of said payload or
vector is an origin of replication, the replication of which depends upon the
presence of a given
protein, peptid, nucleic acid, RNA, molecule or any combination thereof, said
donor bacterial cell
expresses said protein, peptid, nucleic acid, RNA, molecule or any combination
thereof.
[183] Preferably, said protein, peptid, nucleic acid, RNA, molecule or any
combination thereof
is expressed in trans, as defined above.
[184] In a particular embodiment, said donor bacterial cell stably comprises a
nucleic acid
encoding said protein, peptid, nucleic acid, RNA, molecule or any combination
thereof.
[185] In a particular embodiment, when said origin of replication is derived
from phage-
inducible chromosomal islands (PICIs), said conditional origin of replication
is active in said
donor bacterial cell because said donor bacterial cell expresses a rep
protein, in particular a
primase-helicase, in particular a primase-helicase of sequence SEQ ID NO: 50.
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[186] In a particular embodiment, said donor bacterial cell stably comprises a
nucleic acid
encoding said rep protein, in particular said primase-helicase, said nucleic
acid typically
comprising or consisting of the sequence SEQ ID NO: 51.
[187] In a particular embodiment, said donor bacterial cell is a production
cell line, in particular
a cell line producing packaged phagemids including the payload or vector of
the invention.
[188] The delivered nucleic acid of interest preferably comprises a nucleic
acid sequence
under the control of a promoter. In certain embodiments, the nucleic acid of
interest is selected
from the group consisting of a Cas nuclease gene, a Cas9 nuclease gene, a
guide RNA, a
CRISPR locus, a toxin gene, a gene expressing an enzyme such as a nuclease or
a kinase, a
TALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, a membrane
protein, a
structural protein, a secreted protein, a gene expressing resistance to an
antibiotic or to a drug
in general, a gene expressing a toxic protein or a toxic factor, and a gene
expressing a virulence
protein or a virulence factor, and any of their combination. In an embodiment,
the nucleic acid
payload encodes a therapeutic protein. In another embodiment, the nucleic acid
payload
encodes an antisense nucleic acid molecule.
[189] In one embodiment, the sequence of interest is a programmable nuclease
circuit to be
delivered to the targeted bacteria. This programmable nuclease circuit is able
to mediate in vivo
sequence-specific elimination of bacteria that contain a target gene of
interest (e.g. a gene that
is harmful to humans). Some embodiments of the present disclosure relate to
engineered
variants of the Type ll CRISPR-Cas (Clustered Regularly Interspaced Short
Palindromic
Repeats-CRISPR-associated) system of Streptococcus pyogenes. Other
programmable
nucleases that can be used include other CRISPR-Cas systems, engineered TALEN
(Transcription Activator-Like Effector Nuclease) variants, engineered zinc
finger nuclease (ZFN)
variants, natural, evolved or engineered meganuclease or recombinase variants,
and any
combination or hybrids of programmable nucleases. Thus, the engineered
autonomously
distributed nuclease circuits provided herein may be used to selectively
cleave DNA encoding a
gene of interest such as, for example, a toxin gene, a virulence factor gene,
an antibiotic
resistance gene, a remodeling gene or a modulatory gene (cf. W02014124226).
[190] Other sequences of interest, such as programmable sequences, can be
added to the
delivered nucleic acid sequence so as to be delivered to targeted bacteria. In
an embodiment,
the sequence of interest added to the delivered nucleic acid sequence leads to
cell death of the
targeted bacteria. For example, the nucleic acid sequence of interest added to
the plasmid may
encode holins or toxins.
[191] Alternatively, the sequence of interest circuit added to the delivered
nucleic acid
sequence does not lead to bacteria death. For example, the sequence of
interest may encode
reporter genes leading to a luminescence or fluorescence signal.
Alternatively, the sequence of
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interest may comprise proteins and enzymes achieving a useful function such as
modifying the
metabolism of the bacteria or the composition of its environment.
[192] In a particular embodiment, the nucleic acid of interest is selected
from the group
consisting of Cas9, a single guide RNA (sgRNA), a CRISPR locus, a gene
expressing an
enzyme such as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, a
recombinase, a
bacterial receptor, a membrane protein, a structural protein, a secreted
protein, resistance to an
antibiotic or to a drug in general, a gene expressing a toxic protein or a
toxic factor and a gene
expressing a virulence protein or a virulence factor and any of their
combination.
[193] In a particular embodiment, the nucleic acid of interest is a gene
expressing a nuclease.
More particularly, the nuclease may target cleavage of a host bacterial cell
chromosome or a
host bacterial cell plasmid. In a more particular embodiment, the cleavage may
occur in an
antibiotic resistant gene.
[194] In a particular embodiment, the delivered nucleic acid sequence
according to the
disclosure comprises a nucleic acid sequence of interest that encodes a
bacteriocin, which can
be a proteinaceous toxin produced by bacteria to kill or inhibit growth of
other bacteria.
Bacteriocins are categorized in several ways, including producing strain,
common resistance
mechanisms, and mechanism of killing. Such bacteriocin had been described from
gram
negative bacteria (e.g. microcins, colicin-like bacteriocins and tailocins)
and from gram positive
bacteria (e.g. Class I, Class II, Class III or Class IV bacteriocins).
[195] In one embodiment, the delivered nucleic acid sequence according to the
disclosure
further comprises a sequence of interest encoding a toxin selected in the
group consisting of
microcins, colicin-like bacteriocins, tailocins, Class I, Class II, Class III
and Class IV bacteriocins.
[196] In a particular embodiment, the corresponding immunity polypeptide (i.e.
anti-toxin) may
be used to protect bacterial cells (see review by Cotter et al., Nature
Reviews Microbiology 11:
95, 2013, which is hereby incorporated by reference in its entirety) for
delivered nucleic acid
sequence production and encapsidation purpose but is absent in the
pharmaceutical
composition and in the targeted bacteria in which the delivered nucleic acid
sequence of the
disclosure is delivered.
[197] In one aspect of the disclosure, the CRISPR system is included in the
delivered nucleic
acid sequence. The CRISPR system contains two distinct elements, i.e. i) an
endonuclease, in
this case the CRISPR associated nuclease (Cas or "CRISPR associated protein")
and ii) a guide
RNA. The guide RNA is in the form of a chimeric RNA which consists of the
combination of a
CRISPR (RNAcr) bacterial RNA and a RNAtracr (trans-activating RNA CRISPR)
(Jinek et al.,
Science 2012). The guide RNA combines the targeting specificity of the RNAcr
corresponding
to the "spacing sequences" that serve as guides to the Cas proteins, and the
conformational
properties of the RNAtracr in a single transcript. When the guide RNA and the
Cas protein are
expressed simultaneously in the cell, the target genomic sequence can be
permanently modified
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or interrupted. The modification is advantageously guided by a repair matrix.
In general, the
CRISPR system includes two main classes depending on the nuclease mechanism of
action.
Class 1 is made of multi-subunit effector complexes and includes type 1, Ill
and IV. Class 2 is
made of single-unit effector modules, like Cas9 nuclease, and includes type 11
(II-A,11-13,11-C,11-C
variant), V (V-A,V-B,V-C,V-D,V-E,V-U1,V-U2,V-U3,V-U4,V-U5) and VI (VI-A,V1-
131,V1-132,VI-
C,VI-D).
[198] The sequence of interest according to the present disclosure comprises a
nucleic acid
sequence encoding Cas protein. A variety of CRISPR enzymes are available for
use as a
sequence of interest on the plasmid. In some embodiments, the CRISPR enzyme is
a Type 11
CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage.
In
some other embodiments, the CRISPR enzyme catalyzes RNA cleavage. In one
embodiment,
the CRISPR enzymes may be coupled to a sgRNA. In certain embodiments, the
sgRNA targets
a gene selected in the group consisting of an antibiotic resistance gene,
virulence protein or
factor gene, toxin protein or factor gene, a bacterial receptor gene, a
membrane protein gene, a
structural protein gene, a secreted protein gene and a gene expressing
resistance to a drug in
general.
[199] Non-limiting examples of Cas proteins as part of a multi-subunit
effector or as a single-
unit effector include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,
Cas9 (also
known as Csn1 and Csx12), Cas10, Cash 1 (SS), Cas12a (Cpf1), Cas12b (C2c1),
Cas12c
(C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a
(C2c2),
Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc1, Csc2, Cse1, Cse2, Csy1,
Csy2, Csy3,
Csf1, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,
Cmr6,
Csn2, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx13, Csx1, Csx15,
SdCpf1,
CmtCpf1, TsCpf1, CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcCpf1, AsCpf1, LbCpf1,
Mad4, Mad7,
Cms1, homologues thereof, orthologues thereof, variants thereof, or modified
versions thereof.
In some embodiments, the CRISPR enzyme cleaves both strands of the target
nucleic acid at
the Protospacer Adjacent Motif (PAM) site. In a particular embodiment, said
Cas protein is
Cas12a (Cpf1).
[200] In a particular embodiment, the CRISPR enzyme is any Cas9 protein, for
instance any
naturally occurring bacterial Cas9 as well as any variants, homologs or
orthologs thereof.
[201] By "Cas9" is meant a protein Cas9 (also called Csn1 or Csx12) or a
functional protein,
peptide or polypeptide fragment thereof, i.e. capable of interacting with the
guide RNA(s) and of
exerting the enzymatic activity (nuclease) which allows it to perform the
double-strand cleavage
of the DNA of the target genome. "Cas9" can thus denote a modified protein,
for example
truncated to remove domains of the protein that are not essential for the
predefined functions of
the protein, in particular the domains that are not necessary for interaction
with the gRNA(s).
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[202] The sequence encoding Cas9 (the entire protein or a fragment thereof) as
used in the
context of the disclosure can be obtained from any known Cas9 protein (Fonfara
et al., Nucleic
Acids Res 42 (4), 2014; Koonin et al., Nat Rev Microbiol 15(3), 2017).
Examples of Cas9
proteins useful in the present disclosure include, but are not limited to,
Cas9 proteins of
Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1Cas9,
St3Cas9),
Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni
(CjCas9),
Francisella novicida (FnCas9) and Neisseria meningitides (NmCas9).
[203] The sequence encoding Cpf1 (Cas12a) (the entire protein or a fragment
thereof) as used
in the context of the disclosure can be obtained from any known Cpf1 (Cas12a)
protein (Koonin
et al., 2017). Examples of Cpf1(Cas12a) proteins useful in the present
disclosure include, but
are not limited to, Cpf1(Cas12a) proteins of Acidaminococcus sp,
Lachnospiraceae bacteriu and
Francisella novicida.
[204] The sequence encoding Cas13a (the entire protein or a fragment thereof)
can be
obtained from any known Cas13a (C2c2) protein (Abudayyeh et al., 2017).
Examples of Cas13a
(C2c2) proteins useful in the present disclosure include, but are not limited
to, Cas13a (C2c2)
proteins of Leptotrichia wadei (LwaCas13a).
[205] The sequence encoding Cas13d (the entire protein or a fragment thereof)
can be
obtained from any known Cas13d protein (Yan et al., 2018). Examples of Cas13d
proteins useful
in the present disclosure include, but are not limited to, Cas13d proteins of
Eubacterium siraeum
and Ruminococcus sp.
[206] The sequence encoding Mad4 (the entire protein or a fragment thereof) as
used in the
context of the invention is disclosed in international application
W02018/236548.
[207] The sequence encoding Mad7 (the entire protein or a fragment thereof) as
used in the
context of the invention is disclosed in international application
W02018/236548.
[208] The sequence encoding Cms1 (the entire protein or a fragment thereof) as
used in the
context of the invention is disclosed in international patent application
W02017/141173.
[209] In a particular embodiment, the nucleic sequence of interest is a
CRISPR/cas, in
particular a CRISPR/Cas9, system for the reduction of gene expression or
inactivation a gene
selected in the group consisting of an antibiotic resistance gene, virulence
factor or protein gene,
toxin factor or protein gene, a gene expressing a bacterial receptor, a
membrane protein, a
structural protein, a secreted protein, and a gene expressing resistance to a
drug in general.
[210] In one embodiment, the CRISPR system is used to target and inactivate a
virulence
factor. A virulence factor can be any substance produced by a pathogen that
alters host-
pathogen interaction by increasing the degree of damage done to the host.
Virulence factors are
used by pathogens in many ways, including, for example, in cell adhesion or
colonization of a
niche in the host, to evade the host's immune response, to facilitate entry to
and egress from
host cells, to obtain nutrition from the host, or to inhibit other
physiological processes in the host.
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Virulence factors can include enzymes, endotoxins, adhesion factors, motility
factors, factors
involved in complement evasion, and factors that promote biofilm formation.
For example, such
targeted virulence factor gene can be E. coli virulence factor gene such as,
without limitation,
EHEC-HlyA, Stx1 (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c),
Stx2d (VT2d),
Stx2e (VT2e) and Stx2f (VT2f), Stx2h (VT2h), fimA, fimF, fimH, neuC, kpsE,
sfa, foc, iroN, aer,
iha, papC, papGI, papGII, papGIII, hlyC, cnf1, hra, sat, ireA, usp ompT, ibeA,
malX, fyuA, irp2,
traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX, SHV,
csgA, csgB, csgC,
csgD, csgE, csgF, csgG, csgH, T1SS, T2SS, T3SS, T4SS, T5SS, T6SS (secretion
systems).
For example, such targeted virulence factor gene can be Shigella dysenteriae
virulence factor
gene such as, without limitation, stx1 and stx2. For example, such targeted
virulence factor gene
can be Yersinia pestis virulence factor gene such as, without limitation, yscF
(plasmid-borne
(pCDI) T3SS external needle subunit). For example, such targeted virulence
factor gene can be
Francisella tularensis virulence factor gene such as, without limitation,
fsIA. For example, such
targeted virulence factor gene can be Bacillus anthracis virulence factor gene
such as, without
limitation, pag (Anthrax toxin, cell-binding protective antigen). For example,
such targeted
virulence factor gene can be Vibrio cholera virulence factor gene such as,
without limitation, ctxA
and ctx6 (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master
virulence regulator).
For example, such targeted virulence factor gene can be Pseudomonas aeruginosa
virulence
factor genes such as, without limitation, pyoverdine (e.g., sigma factor pvdS,
biosynthetic genes
pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvd0, pvdP, transporter
genes pvdE,
pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE,
pchF and
pchG, and toxins (e.g., exoU, exoS and exoT). For example, such targeted
virulence factor gene
can be Klebsiella pneumoniae virulence factor genes such as, without
limitation, fimA
(adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
For example,
such targeted virulence factor gene can be Acinetobacter baumanniivirulence
factor genes such
as, without limitation, ptk (capsule polymerization) and epsA (assembly). For
example, such
targeted virulence factor gene can be Salmonella enterica Typhi virulence
factor genes such as,
without limitation, MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator),
and those associated
with bile tolerance, including efflux pump genes acrA, acrB and toIC. For
example, such targeted
virulence factor gene can be Fusobacterium nucleatum virulence factor genes
such as, without
limitation, FadA and TIGIT. For example, such targeted virulence factor gene
can be Bacteroides
fragilis virulence factor genes such as, without limitation, bft.
[211] In another embodiment, the CRISPR/Cas system is used to target and
inactivate an
antibiotic resistance gene such as, without limitation, GyrB, ParE, ParY,
AAC(1), AAC(2'),
AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"),
APH(3'),
APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-
M, KPC,
SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA 13-
lactamase,
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mecA, 0mp36, OmpF, PIB, bla (blal, blaR1) and mec (mecl, mecR1) operons,
Chloramphenicol
acetyltransf erase (CAT), Chloramphenicol phosphotransferase,
Ethambutol-resistant
arabinosyltransferase (EmbB), MupA, MupB, Integral membrane protein MprF, Cfr
23S rRNA
methyltransf erase, Rifampin ADP-ribosyltransferase (Arr), Rifampin
glycosyltransf erase,
Rifampin monooxygenase, Rifampin phosphotransferase, DnaA, RbpA, Rifampin-
resistant beta-
subunit of RNA polymerase (RpoB), Erm 23S rRNA methyltransferases, Lsa, MsrA,
Vga, VgaB,
Streptogramin Vgb lyase, Vat acetyltransf erase, Fluoroquinolone acetyltransf
erase,
Fluoroquinolone-resistant DNA topoisomerases, Fluoroquinolone-resistant GyrA,
GyrB, ParC,
Quinolone resistance protein (Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA,
VanB, VanD,
VanR, VanS, Lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt,
Ole, Macrolide
phosphotransferases (MPH), MefA, MefE, Mel, Streptothricin acetyltransferase
(sat), Sul1, 5u12,
5u13, sulfonamide-resistant FolP, Tetracycline inactivation enzyme TetX, TetA,
TetB, TetC,
Tet30, Tet31, TetM, Tet0, TetQ, Tet32, Tet36, MacAB-ToIC, MsbA, MsrA,VgaB,
EmrD, EmrAB-
To1C, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE, adeR, acrR,
baeSR,
mexR, phoPQ, mtrR, or any antibiotic resistance gene described in the
Comprehensive
Antibiotic Resistance Database (CARD https://card.mcmaster.ca/).
[212] In another embodiment, the CRISPR/Cas system is used to target and
inactivate a
bacterial toxin gene. Bacterial toxins can be classified as either exotoxins
or endotoxins.
Exotoxins are generated and actively secreted; endotoxins remain part of the
bacteria. The
response to a bacterial toxin can involve severe inflammation and can lead to
sepsis. Such toxin
can be for example Botulinum neurotoxin, Tetanus toxin, Staphylococus toxins,
Diphteria toxin,
Anthrax toxin, Alpha toxin, Pertussis toxin, Shiga toxin, Heat-stable
enterotoxin (E. coil ST),
colibactin, BFT (B. fragilis toxin) or any toxin described in Henkel et al.,
(Toxins from Bacteria in
EXS. 2010; 100: 1-29). In a particular embodiment, said toxin is Shiga toxin.
[213] In another embodiment, the nucleic acid of interest encodes a gene or
group of genes
encoding one or more exogenous enzyme(s) which result(s) in a genetic
modification.
[214] In a particular embodiment, said nucleic acid of interest is a gene
encoding a base-editor
or a prime-editor.
[215] In some embodiments, the genetic modification is made with one or more
of the following
enzymes and systems.
[216] Cytosine base editors (CBE) and Adenosine base editors (ABE), as
described in Rees
et al. (2018) Nat Rev Genet 19:770-788, which is hereby incorporated by
reference.
[217] So far there is seven types of DNA base editors described:
= Cytosine Base Editor (CBE) that convert C:G into T:A (Komor et al. (2016)
Nature 533:420-
424)
= Adenine Base Editor (ABE) that convert A:T into G:C (Gaudelli et aL
(2017) Nature
551 :464-471)
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= Cytosine Guanine Base Editor (CGBE) that convert C:G into G:C (Chen et
al. (2020)
Biorxiv "Precise and programmable C:G to G:C base editing in genomic DNA";
Kurt etal.
(2020) Nat. Biotechnol. "CRISPR C-to-G base editors for inducing targeted DNA
transversions in human cells")
= Cytosine Adenine Base Editor (CABE) that convert C:G into A:T (Zhao etal.
(2020) Nature
Biotechnol. "New base editors change C to A in bacteria and C to G in
mammalian cells")
= Adenine Cytosine Base Editor (ACBE) that convert A:T into C:G
(W02020181180)
= Adenine Thymine Base Editor (ATBE) that convert A:T into T:A
(W02020181202)
= Thymine Adenine Base Editor (TABE) that convert T:A into A:T
(W02020181193,
W02020181178, W02020181195)
[218] Base editors differ in the base modification enzymes. CBE rely on ssDNA
cytidine
deaminase among which: APOBEC1, rAPOBEC1, APOBEC1 mutant or evolved version
(evoAPOBEC1), and APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine
deaminase 1
(CDA1), evoCDA1, FERNY, evoFERNY.
[219] ABE rely on deoxyadenosine deaminase activity of a tandem fusion TadA-
TadA* where
TadA* is an evolved version of TadA, an E.coli tRNA adenosine deaminase
enzyme, able to
convert adenosine into lnosine on ssDNA.TadA* include TadA-8a-e and TadA-7.10.
[220] Except from base modification enzyme there has been also modifications
implemented
to base editor to increase editing efficacy, precision and modularity:
= the addition of one or two uracil DNA glycosylase inhibitor domain (UGI)
to prevent base
excision repair mechanism to revert base edition
= the addition of Mu-GAM that decrease insertion-deletion rate by
inhibiting Non-
homologous end joining mechanism in the cell (NHEJ)
= the use of nickase active Cas9 (nCas9 D10A) that, by creating nicks on
the non-edited
strand favor its repair and consequently the fixation of the edited base
= the use of diverse Cas proteins from for example different organisms,
mutants with different
PAM motifs or different fidelity or different family (e.g. Cas12a).
[221] Non-limiting examples of DNA based editor proteins include BD , BE2,
BE3, BE4, BE4-
GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-
BE3,
YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3,
EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10,
ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY-
CBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4,
enAsBE1.1, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBE1.1, AsBE1.4, CRISPR-Abest,
CRISPR-Cbest, eA3A-BE3, AncBE4.
[222] Cytosine Guanine Base Editors (CGBE) consist of a nickase CRISPR fused
to:
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[a] A cytosine deaminase (rAPOBEC) and base excision repair proteins (e.g.
rXRCC1)
(Chen et al. (2020) Biorxiv "Precise and programmable C:G to G:C base editing
in
genomic DNA").
[b] A rat APOBEC1 variant (R33A) protein and an E. co/i-derived uracil DNA N-
glycosylase
(eUNG) (Kurt et aL (2020) Nat. BiotechnoL "CRISPR C-to-G base editors for
inducing
targeted DNA transversions in human cells").
[223] Cytosine Adenine Base Editors (CABE) consist of a Cas9 nickase, a
cytidine deaminase
(e.g. AID), and a uracil-DNA glycosylase (Ung) (Zhao et aL (2020) Nature
BiotechnoL "New base
editors change C to A in bacteria and C to G in mammalian cells").
[224] ACBE include a nucleic acid programmable DNA-binding protein and an
adenine oxidase
(W02020181180).
[225] ATBE consist of a Cas9 nickase and one or more adenosine deaminase or an
oxidase
domain (W02020181202).
[226] TABE consist of a Cas9 nickase and an adenosine methyltransferase, a
thymine
alkyltransferase, or an adenosine deaminase domain (W02020181193,
W02020181178,
W02020181195).
[227] Base editor molecules can also consist of two or more of the above
listed editor enzymes
fused to a Cas protein (e.g. combination of an ABE and CBE). These
biomolecules are named
dual base editors and enable the editing of two different bases (Grunewald et
aL (2020) Nature
BiotechnoL "A dual-deaminase CRISPR base editor enables concurrent adenine and
cytosine
editing"; Li et aL (2020) Nature BiotechnoL "Targeted, random mutagenesis of
plant genes with
dual cytosine and adenine base editors").
[228] Prime editors (PE), as described in Anzalone etal. (2019) Nature 576:149-
157, which
is hereby incorporated by reference, consist of nCas9 fused to a reverse
transcriptase used in
combination with a prime editing RNA (pegRNA, a guide RNA that includes a
template region
for reverse transcription).
[229] Prime Editing allows introduction of insertions, deletions (indels) and
12 base-to-base
conversions. Prime editing relies on the ability of a reverse transcriptase
(RT), fused to a Cas
nickase variant, to convert RNA sequence brought by a prime editing guide RNA
(peg RNA) into
DNA at the nick site generated by the Cas protein. The DNA flap generated from
this process is
then included or not in the targeted DNA sequence.
[230] Prime editing systems include:
= a Cas nickase variant such as Cas9-H840A fused to a reverse transcriptase
domain such
as M-MLV RT or its mutant version (M-MLV RT(D200N), M-MLV RT(D200N/L603W), M-
MLV RT(D200N/L603W/T330P/ T306K/VV313F)
= a prime editing guide RNA (pegRNA)
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[231] To favor editing the prime editing system can include the expression of
an additional
sgRNA targeting the Cas nickase activity towards the non-edited DNA strand
ideally only after
the resolution of the edited strand flap by designing the sgRNA to anneal with
the edited strand
but not with the original strand.
[232] Non-limiting examples of prime editing systems include PE1, PE1-M1, PE1-
M2, PE1-
M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.
[233] Cas9 Retron precISe Parallel Editing via homologY (`CRISPEY'), a retron
RNA fused to
the sgRNA and expressed together with Cas9 and the retron proteins including
at least the
reverse transcriptase (Sharon etal. (2018) Cell 175:544-557.e16).
[234] The SCRIBE strategy: a retron system expressed in combination with a
recombinase
promoting the recombination of single stranded DNA, also known as single
stranded annealing
proteins (SSAPs) (Farzadfard & Lu (2014) Science 346:1256272). Such
recombinases include
but are not limited to phage recombinases such as lambda red, recET, Sak,
5ak4, and newly
described SSAPs described in Wannier et al. (2020) Proc Nat! Acad Sci U S A
117(24):13689-
13698 which is hereby incorporated by reference.
[235] The targetron system based on group ll introns described in Karberg et
aL (2001) Nat
Biotechnol 19:1162-7, which is hereby incorporated by reference, and which has
been adapted
to many bacterial species.
[236] Other retron based gene targeting approaches are described in Simon et
al. (2019)
Nucleic Acids Res 47:11007-11019, which is hereby incorporated by reference.
[237] In various embodiments, the nucleic acid of interest encodes fusion
proteins comprising
a Cas, in particular Cas9 (e.g., a Cas9 nickase), domain and a deaminase
domain. In some
embodiments, the fusion protein comprises a Cas, in particular Cas9, and a
cytosine deaminase
enzyme, such as APOBEC enzymes, or adenosine deaminase enzymes, such as ADAT
enzymes, for example as disclosed in U.S. Patent Publ. 2015/0166980, which is
hereby
incorporated by reference. In one embodiment, the deaminase is an ACF1/ASE
deaminase.
[238] In various embodiments, the APOBEC deaminase is selected from the group
consisting
of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B
deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase,
APOBEC3G deaminase, and APOBEC3H deaminase. In various embodiments, the fusion
protein comprises a Cas9 domain, a cytosine deaminase domain, and a uracil
glycosylase
inhibitor (UGI) domain.
[239] In one embodiment, the deaminase is an adenosine deaminase that
deaminate
adenosine in DNA, for example as disclosed in U.S. Patent 10,113,163, which is
hereby
incorporated by reference. In some embodiments, the fusion proteins further
comprise an
inhibitor of base repair, such as, a nuclease dead inosine specific nuclease
(dISN), for example
as disclosed in U.S. Patent 10,113,163. In various embodiments, the nucleic
acid of interest
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encodes fusion proteins comprising a catalytically impaired Cas, in particular
Cas9,
endonuclease fused to an engineered reverse transcriptase, programmed with a
prime editing
guide RNA (pegRNA) that both specifies the target site and encodes the desired
edit, for
example as described in Anzalone et al. (2019) Nature 576:149-157, which is
hereby
incorporated by reference.
[240] In some embodiments, the genetic modification is made at the RNA level.
RNA base
editing is based on the same principle as DNA base editing: an enzyme
catalyzing the
conversion of a RNA base into another must be brought close to the target base
to perform its
conversion locally. In one embodiment, the enzyme used for RNA editing is an
adenosine
deaminase from ADAR family that converts Adenosine into lnosine in dsRNA
structure. Several
seminal studies used this specificity for dsRNA and fused the ADAR deaminase
domain
(ADARDD) to an antisense oligo in order to program local RNA base editing.
More recently the
ability of some CRISPR-Cas systems to bind RNA molecules was repurposed into
RNA editing.
Using catalytically dead Cas13b enzyme (dPspCas13b) fused to a hyperactive
mutant of ADAR2
deaminase domain (ADAR2DD-E4880 for REPAIRv1 and ADAR2DD-E4880-1375G for
REPAIRv2) Cox et al improved specificity and efficiency compare to previous
RNA editing
strategies. Non-limiting examples of RNA based editor proteins include
REPAIRv1, REPAIRv2.
[241] In some embodiments, the nucleic acid of interest encodes other
programmable
nucleases. These include an engineered TALEN (Transcription Activator-Like
Effector
Nuclease) and variants, engineered zinc finger nuclease (ZFN) variants,
natural, evolved or
engineered meganuclease or recombinase variants, and any combination or
hybrids of
programmable nucleases. Thus, the programmable nucleases provided herein may
be used to
selectively modify DNA encoding a gene of interest such as, for example, a
toxin gene, a
virulence factor gene, an antibiotic resistance gene, a remodeling gene or a
modulatory gene
(cf. W02014124226 and U52015/0064138).
[242] In a particular embodiment, said payload comprises or consists of the
nucleic acid
sequence SEQ ID NO: 33. In an alternative embodiment, said payload comprises
or consists of
the nucleic acid sequence SEQ ID NO: 42.
[243] In an alternative embodiment, the nucleic acid of interest encodes a
therapeutic protein.
In another embodiment, the nucleic acid of interest encodes an antisense
nucleic acid molecule.
[244] The present disclosure thus also provides a production cell line, as
defined above,
comprising a helper prophage as defined above, and further comprising a
phagemid comprising
or consisting of the payload as defined above, in particular of the nucleic
acid sequence SEQ ID
NO: 33 or of the nucleic acid sequence SEQ ID NO: 42.
[245] In a particular embodiment, the bacterial delivery vehicle provided
herein comprises
chimeric STF of sequence SEQ ID NO: 11 and chimeric gpJ variant of sequence
SEQ ID NO:
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27, and further comprises a payload which comprises or consists of the nucleic
acid sequence
SEQ ID NO: 33.
[246] In another particular embodiment, the bacterial delivery vehicle
provided herein
comprises chimeric STF of sequence SEQ ID NO: 11 and chimeric gpJ variant of
sequence SEQ
ID NO: 27, and further comprises a payload which comprises or consists of the
nucleic acid
sequence SEQ ID NO: 42.
Targeted bacteria
[247] The bacteria targeted by bacterial delivery vehicles disclosed herein
can be any bacteria
present in a mammal organism. In a certain aspect, the bacteria are targeted
through interaction
of the chimeric RBPs of the delivery vehicles with the bacterial cell. It can
be any commensal,
symbiotic or pathogenic bacteria of the microbiota or microbiome.
[248] A microbiome may comprise a variety of endogenous bacterial species, any
of which
may be targeted in accordance with the present disclosure. In some
embodiments, the genus
and/or species of targeted endogenous bacterial cells may depend on the type
of
bacteriophages being used for preparing the bacterial delivery vehicles. For
example, some
bacteriophages exhibit tropism for, or preferentially target, specific host
species of bacteria.
Other bacteriophages do not exhibit such tropism and may be used to target a
number of
different genus and/or species of endogenous bacterial cells.
[249] Examples of bacterial cells include, without limitation, cells from
bacteria of the genus
Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp.,
Bordetella spp., Neisseria
spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter
spp., Chlamydia
spp., Hemophilus spp., Bruce/la spp., Mycobacterium spp., Legionella spp.,
Rhodococcus spp.,
Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp.,
Erysipelothrix spp., Salmonella
spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacteroides
spp., Prevotella
spp., Clostridium spp., Bifidobacterium spp., Clostridium spp., Brevibacterium
spp., Lactococcus
spp., Leuconostoc spp., Actinobacillus spp., Selnomonas spp., Shigella spp.,
Zymonas spp.,
Mycoplasma spp., Treponema spp., Leuconostoc spp., Corynebacterium spp.,
Enterococcus
spp., Enterobacter spp., Pyrococcus spp., Serratia spp., Morganella spp.,
Parvimonas spp.,
Fusobacterium spp., Actinomyces spp., Porphyromonas spp., Micrococcus spp.,
Bartonella
spp., Borrelia spp., Brucelia spp., Campylobacter spp., Chlamydophilia spp.,
Cutibacterium
(formerly Propionibacterium) spp., Ehrlichia spp., Haemophilus spp.,
Leptospira spp., Listeria
spp., Mycoplasma spp., Nocardia spp., Rickettsia spp., Ureaplasma spp., and
Lactobacillus spp,
and a mixture thereof.
[250] Thus, bacterial delivery vehicles may target (e.g., specifically target)
a bacterial cell from
any one or more of the foregoing genus of bacteria to specifically deliver the
payload of interest
according to the disclosure.
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[251] In an embodiment, the targeted bacteria can be selected from the group
consisting of
Y ersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp.,
Pseudomonas spp.,
Helicobacter spp., Vibrio spp, Salmonella spp., Streptococcus spp.,
Staphylococcus spp.,
Bacteroides spp., Clostridium spp., Shigella spp., Enterococcus spp.,
Enterobacter spp., and
Listeria spp.
[252] In some embodiments, targeted bacterial cells of the present disclosure
are anaerobic
bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic
bacterial cells include
facultative anaerobic cells such as but not limited to Escherichia coil,
Shewanella oneidensis
and Listeria. Anaerobic bacterial cells also include obligate anaerobic cells
such as, for example,
Bacteroides and Clostridium species. In humans, anaerobic bacteria are most
commonly found
in the gastrointestinal tract. In some particular embodiment, the targeted
bacteria are thus
bacteria most commonly found in the gastrointestinal tract. Bacteriophages
used for preparing
the bacterial virus particles, and then the bacterial virus particles, may
target (e.g., to specifically
target) anaerobic bacterial cells according to their specific spectra known by
the person skilled
in the art to specifically deliver the plasmid.
[253] In some embodiments, the targeted bacterial cells are, without
limitation, Bacteroides
thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides
vulgatus, Clostridium
leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis,
Clostridium butyricum,
Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis,
Leuconostoc
lactis, Actinobacillus actinomycetemcomitans, cyanobacteria, Escherichia coil,
Helicobacter
pylori, Selenomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma
mycoides,
Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis,
Leuconostoc oenos,
Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus,
Lactobacillus
casei, Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans,
Bacillus cereus,
Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens,
Pyrococcus abyssi,
Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus,
Lactobacillus
pentosus, Bacteroides Ira gills, Staphylococcus epidermidis, Streptomyces
phaechromo genes,
Streptomyces ghanaenis, Klebsiella pneumoniae, Enterobacter cloacae,
Enterobacter
aero genes, Serratia marcescens, Morganella morganii, Citrobacter freundii,
Pseudomonas
aeruginosa, Parvimonas micra, Prevotella intermedia, Fusobacterium nucleatum,
Prevotella
nigrescens, Actinomyces israelii, Porphyromonas endodontalis, Porphyromonas
gin givalis
Micrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonas
caviae, Bacillus
anthracis, Bartonella henselae, Bartonella Quintana, Bordetella pertussis,
Borrelia burgdorferi,
Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus,
Brucella canis, Brucella
melitensis, Brucella suis, Campylobacter jejuni, Campylobacter coil,
Campylobacter fetus,
Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci,
Clostridium botulinum,
Clostridium difficile, Clostridium perfringens, Clostridium tetani,
Corynebacterium diphtheria,
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Cutibacterium acnes (formerly Propionibacterium acnes), Ehrlichia canis,
Ehrlichia chaffeensis,
Enterococcus faecium, Francisella tularensis, Haemophilus influenza,
Legionella pneumophila,
Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira
noguchii, Listeria
monocyto genes, Mycobacterium leprae, Mycobacterium tuberculosis,
Mycobacterium ulcerans,
Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Nocardia
asteroids,
Rickettsia rickettsia, Salmonella enteritidis, Salmonella typhi, Salmonella
paratyphi, Salmonella
typhimurium, Shigella flexnerii, Shigella dysenteriae, Staphylococcus
saprophyticus,
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans,
Treponema
pallidum, Ureaplasma urealyticum, Vibrio cholera, Vibrio parahaemolyticus,
Yersinia pestis,
Yersinia enterocolitica, Yersinia pseudotuberculosis, Actinobacter baumanii,
Pseudomonas
aeruginosa, and a mixture thereof. In an embodiment the targeted bacteria of
interest are
selected from the group consisting of Escherichia coil, Enterococcus faecium,
Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa,
Enterobacter cloacae, and Enterobacter aerogenes, and a mixture thereof.
[254] In some embodiments, the targeted bacterial cells are, without
limitation, Anaerotruncus,
Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum,
Anaerosinus,
Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus,
Dehalobacter, Dialister,
Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium,
Gracilibacter,
Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus,
Leuconostoc,
Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter,
Proprionispira,
Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina,
Seinonella,
Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum,
Syntrophococcus, Thermobacillus, Turibacter, WeiseIla, Clostridium,
Bacteroides,
Ruminococcus, Faecalibacterium, Treponema, Phascolarctobacterium, Megasphaera,
Faecalibacterium, Bifidobacterium, Lactobacillus, Sutterella, and/or
Prevotella.
[255] In other embodiments, the targeted bacteria cells are, without
limitation, Achromobacter
xylosoxidans, Acidaminococcus fermentans, Acidaminococcus intestini,
Acidaminococcus sp.,
Acinetobacter baumannii, Acinetobacter junii, Acinetobacter Iwo ffii,
Actinobacillus capsulatus,
Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus,
Actinomyces radingae,
Adlercreutzia equolifaciens, Aeromicrobium massiliense,
Aggregatibacter
actinomycetemcomitans, Akkermansia muciniphila, Aliagarivorans marinus,
Alistipes finegoldii,
Alistipes indistinctus, Alistipes mops, Alistipes onderdonkii, Alistipes
putredinis, Alistipes
senegalensis, Alistipes shahii, Alistipes timonensis, Alloscardo via
omnicolens, Anaerobacter
polyendosporus, Anaerobaculum hydrogeniformans, Anaerococcus hydrogenalis,
Anaerococcus prevotii, Anaerococcus senegalensis, Anaerofustis
stercorihominis, Anaerostipes
caccae, Anaerostipes hadrus, Anaerotruncus colihominis, Aneurinibacillus
aneurinilyticus,
Bacillus licheniformis, Bacillus massilioanorexius, Bacillus
massiliosenegalensis, Bacillus
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simplex, Bacillus smithii, Bacillus subtilis, Bacillus thuringiensis, Bacillus
timonensis,
Bacteroides xylanisolvens, Bacteroides acidifaciens, Bacteroides caccae,
Bacteroides
capillosus, Bacteroides cellulosilyticus, Bacteroides clarus, Bacteroides
coprocola, Bacteroides
coprophilus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis,
Bacteroides finegoldii,
Bacteroides fluxus, Bacteroides fragilis, Bacteroides gallinarum, Bacteroides
intestinalis,
Bacteroides nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides
pectinophilus,
Bacteroides plebeius, Bacteroides salanitronis, Bacteroides salyersiae,
Bacteroides sp.,
Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis,
Bacteroides
vulgatus, Bacteroides xylanisolvens, Bacteroides pectinophilus ATCC,
Bamesiella
intestinihominis, Bavariicoccus seileri, Bifidobacterium adolescentis,
Bifidobacterium angulatum,
Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve,
Bifidobacterium
catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum,
Bifidobacterium Ion gum,
Bifidobacterium pseudocatenulatum, Bifidobacterium stercoris, Bilophila
wadsworthia, Blautia
faecis, Blautia hansenii, Blautia hydrogenotrophica, Blautia luti, Blautia
obeum, Blautia producta,
Blautia wexlerae, Brachymonas chironomi, Brevibacterium senegalense,
Bryantella
form atexigens, butyrate-producing bacterium, Butyricicoccus pullicaecorum,
Butyricimonas
virosa, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Caldicoprobacter
faecalis,
Campylobacter concisus, Campylobacter jejuni, Campylobacter upsaliensis,
Catenibacterium
mitsuokai, Cedecea davisae, Cellulomonas massiliensis, Cetobacterium somerae,
Citrobacter
braakii, Citrobacter freundii, Citrobacter pasteurii, Citrobacter sp.,
Citrobacter youngae,
Cloacibacillus evryensis, Clostridiales bacterium, Clostridioides difficile,
Clostridium
asparagiforme, Clostridium bartlettii, Clostridium boliviensis, Clostridium
bolteae, Clostridium
hathewayi, Clostridium hiranonis, Clostridium hylemonae, Clostridium leptum,
Clostridium
methylpentosum, Clostridium nexile, Clostridium orbiscindens, Clostridium
ramosum,
Clostridium scindens, Clostridium sp, Clostridium sp., Clostridium spiroforme,
Clostridium
sporo genes, Clostridium symbiosum, Collinsella aerofaciens, Collinsella
intestinalis, Collinsella
stercoris, Collinsella tanakaei, Coprobacillus cateniformis, Coprobacter
fastidiosus,
Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Corynebacterium
ammonia genes, Corynebacterium amycolatum, Corynebacterium
pseudodiphtheriticum,
Cutibacterium acnes, Dermabacter hominis, Desulfitobacterium hafniense,
Desulfovibrio
fairfieldensis, Desulfovibrio piger, Dialister succinatiphilus, Dielma
fastidiosa, Dorea
formicigenerans, Dorea longicatena, Dysgonomonas capnocytophagoides,
Dysgonomonas
gadei, Dysgonomonas mossii, Edwardsiella tarda, Eggerthella lenta,
Eisenbergiella tayi,
Enorma massiliensis, Enterobacter aerogenes, Enterobacter asburiae,
Enterobacter
cancerogenus, Enterobacter cloacae, Enterobacter massiliensis, Enterococcus
casseliflavus,
Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus
flavescens,
Enterococcus gallinarum, Enterococcus sp., Enterovibrio nigricans,
Erysipelatoclostridium
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ramosum, Escherichia coil, Escherichia sp., Eubacterium biforme, Eubacterium
dolichum,
Eubacterium hallii, Eubacterium limosum, Eubacterium ramulus, Eubacterium
rectale,
Eubacterium siraeum, Eubacterium ventriosum, Exiguobacterium marinum,
Exiguobacterium
undae, Faecalibacterium cf, Faecalibacterium prausnitzii, Faecalitalea
cylindroides, Ferrimonas
balearica, Finegoldia magna, Flavobacterium daejeonense, Flavonifractor
plautii,
Fusicatenibacter saccharivorans, Fusobacterium gonidiaformans, Fusobacterium
mortiferum,
Fusobacterium necropho rum, Fusobacterium nucleatum, Fusobacterium
periodonticum,
Fusobacterium sp., Fusobacterium ulcerans, Fusobacterium varium,
Gallibacterium anatis,
Gemmiger formicilis, Gordonibacter pamelaeae, Hafnia alvei, Helicobacter
bills, Helicobacter
bills, Helicobacter canadensis, Helicobacter canis, Helicobacter cinaedi,
Helicobacter macacae,
Helicobacter pametensis, Helicobacter pullorum, Helicobacter pylori,
Helicobacter rodentium,
Helicobacter winghamensis, Herbaspirillum massiliense, Holdemanella biformis,
Holdemania
fdiformis, Holdemania filiformis, Holdemania massiliensis, Holdemania
filiformis, Hungatella
hathewayi, lntestinibacter bartlettii, lntestinimonas butyriciproducens,
Klebsiella oxytoca,
Klebsiella pneumoniae, Kurthia massiliensis, Lachnospira pectinoschiza,
Lactobacillus
acidophilus, Lactobacillus amylolyticus, Lactobacillus anima/is, Lactobacillus
antri, Lactobacillus
brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus curvatus,
Lactobacillus
delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus
helveticus,
Lactobacillus hilgardii, Lactobacillus iners, Lactobacillus intestinalis,
Lactobacillus johnsonii,
Lactobacillus murinus, Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus reuteri,
Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei,
Lactobacillus salivarius,
Lactobacillus ultunensis, Lactobacillus vagina/is, Lactobacillus plantarum
subsp., Leuconostoc
mesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria
innocua,
Mannheimia granulomatis, Marvinbryantia formatexigens, Megamonas funiformis,
Megamonas
hypermegale, Methanobrevibacter smithii, Methanobrevibacter smithii,
Micrococcus luteus,
Micro virgula aerodenitrificans, Mitsuokella jalaludinii, Mitsuokella
multacida, Mollicutes
bacterium, Murimonas intestini, Neisseria macacae, Nitriliruptor alkaliphilus,
Oceanobacillus
massiliensis, Odoribacter laneus, Odoribacter splanchnicus, Omithobacterium
rhinotracheale,
Oxalobacter formigenes, Paenibacillus barengoltzii, Paenibacillus
chitinolyticus, Paenibacillus
lautus, Paenibacillus motobuensis, Paenibacillus senegalensis,
Paenisporosarcina
quisquiliarum, Parabacteroides distasonis, Parabacteroides goldsteinii,
Parabacteroides
gordonii, Parabacteroides johnsonii, Parabacteroides merdae, Paraprevotella
xylaniphila,
Parasutterella excrementihominis, Parvimonas micra, Pediococcus acidilactici,
Peptoclostridium
difficile, Peptoniphilus harei, Peptoniphilus obesi, Peptoniphilus
senegalensis, Peptoniphilus
timonensis, Phascolarctobacterium succinatutens, Porphyromonas
asaccharolytica,
Porphyromonas uenonis, Prevotella baroniae, Prevotella bivia, Prevotella
copri, Prevotella
dentalis, Prevotella micans, Prevotella multisaccharivorax, Prevotella oralis,
Prevotella salivae,
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Prevotella stercorea, Prevotella veroralis, Propionibacterium acnes,
Propionibacterium avidum,
Propionibacterium freudenreichii, Propionimicrobium lymphophilum, Proteus
mirabilis, Proteus
penneri ATCC, Pro videncia alcalifaciens, Pro videncia rettgeri, Pro videncia
rustigianii,
Pro videncia stuartii, Pseudoflavonifractor capillosus, Pseudomonas
aeruginosa, Pseudomonas
luteola, Ralstonia pickettii, Rheinheimera perlucida, Rheinheimera texasensis,
Riemerella
columbina, Romboutsia lituseburensis, Roseburia faecis, Roseburia
intestinalis, Roseburia
inulinivorans, Ruminococcus bicirculans, Ruminococcus bromii, Ruminococcus
callidus,
Ruminococcus champanellensis, Ruminococcus faecis, Ruminococcus gnavus,
Ruminococcus
lactaris, Ruminococcus obeum, Ruminococcus sp, Ruminococcus sp., Ruminococcus
torques,
Sarcina ventriculi, Sellimonas intestinalis, Senegalimassilia anaerobia,
Shigella sonnei, Slackia
piriformis, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus
nepalensis,
Staphylococcus pseudintermedius, Staphylococcus xylosus, Stenotrophomonas
maltophilia,
Streptococcus agalactiae, Streptococcus anginosus, Streptococcus australis,
Streptococcus
caballi, Streptococcus castoreus, Streptococcus didelphis, Streptococcus
equinus,
Streptococcus gordonii, Streptococcus henryi, Streptococcus hyovaginalis,
Streptococcus
infantarius, Streptococcus infantis, Streptococcus lutetiensis, Streptococcus
merionis,
Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus
ovis,
Streptococcus parasanguinis, Streptococcus plurextorum, Streptococcus porci,
Streptococcus
pyo genes, Streptococcus saliva rius, Streptococcus sobrinus, Streptococcus
the rmophilus,
Streptococcus thoraltensis, Streptomyces albus, Subdoligranulum variabile,
Succinatimonas
hippei, Sutterella parviru bra, Sutterella wads worthensis, Terrisporobacter
glycolicus,
Terrisporobacter mayombei, Thalassobacillus devorans, TimoneIla senegalensis,
Turicibacter
sanguinis, unknown sp, unknown sp., Varibaculum cambriense, Veillonella
atypica, Veillonella
dispar, Veillonella parvula, Vibrio cincinnatiensis, Virgibacillus salexigens
or Weissella
paramesenteroides .
[256] In other embodiments, the targeted bacteria cells are those commonly
found on the skin
microbiota and are without limitation Acetobacter farinalis, Acetobacter
malorum, Acetobacter
orleanensis, Acetobacter sicerae, Achromobacter anxifer, Achromobacter
denitrificans,
Achromobacter marplatensis, Achromobacter spanius, Achromobacter xylosoxidans
subsp.
xylosoxidans, Acidovorax konjaci, Acidovorax radicis, Acinetobacter johnsonii,
Actinomadura
citrea, Actinomadura coerulea, Actinomadura fibrosa, Actinomadura fulvescens,
Actinomadura
jiaoheensis, Actinomadura luteofluorescens, Actinomadura mexicana,
Actinomadura
nitritigenes, Actinomadura verrucosospora, Actinomadura yumaensis, Actinomyces
odontolyticus, Actinomycetospora atypica, Actinomycetospora corticicola,
Actinomycetospora
rhizophila, Actinomycetospora rishiriensis, Aeromonas australiensis, Aeromonas
bestiarum,
Aeromonas bivalvium, Aeromonas encheleia, Aeromonas eucrenophila, Aeromonas
hydrophila
subsp. hydrophila, Aeromonas piscicola, Aeromonas popoffii, Aeromonas rivuli,
Aeromonas
CA 03206312 2023-06-22
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salmonicida subsp. pectinolytica, Aeromonas salmonicida subsp. smithia,
Amaricoccus
kaplicensis, Amaricoccus veronensis, Aminobacter aganoensis, Aminobacter
ciceronei,
Aminobacter lissarensis, Aminobacter niigataensis, Ancylobacter polymorphus,
Anoxybacillus
flavithermus subsp. yunnanensis, Aquamicrobium aerolatum, Archangium gephyra,
Archangium
gephyra, Archangium minus, Archangium violaceum, Arthrobacter viscosus,
Bacillus anthracis,
Bacillus australimaris, Bacillus drentensis, Bacillus mycoides, Bacillus
pseudomycoides,
Bacillus pumilus, Bacillus safensis, Bacillus vallismortis, Bosea thiooxidans,
Bradyrhizobium
huanghuaihaiense, Bradyrhizobium japonicum, Brevundimonas aurantiaca,
Brevundimonas
intermedia, Burkholderia aspalathi, Burkholderia choica, Burkholderia
cordobensis, Burkholderia
diffusa, Burkholderia insulsa, Burkholderia rhynchosiae, Burkholderia
terrestris, Burkholderia
udeis, Buttiauxella gaviniae, Caenimonas terrae, Capnocytophaga gingivalis,
Chitinophaga
din ghuensis, Chryseobacterium gleum, Chryseobacterium greenlandense,
Chryseobacterium
jejuense, Chryseobacterium piscium, Chryseobacterium sediminis,
Chryseobacterium tructae,
Chryseobacterium ureilyticum, Chryseobacterium vietnamense, Corynebacterium
accolens,
Corynebacterium afermentans subsp. lipophilum, Corynebacterium minutissimum,
Corynebacterium sundsvallense, Cupriavidus metallidurans, Cupriavidus
nantongensis,
Cupriavidus necator, Cupriavidus pampae, Cupriavidus yeoncheonensis,
Curtobacterium
flaccumfaciens, Devosia epidermidihirudinis, Devosia riboflavina, Devosia
riboflavina,
Diaphorobacter oryzae, Dietzia psychralcaliphila, Ensifer adhaerens, Ensifer
americanus,
Enterococcus malodoratus, Enterococcus pseudoavium, Enterococcus viikkiensis,
Enterococcus xiangfangensis, Erwinia rhapontici, Falsirhodobacter
halotolerans,
Flavobacterium araucananum, Flavobacterium frigidimaris, Gluconobacter
frateurii,
Gluconobacter thailandicus, Gordonia alkanivorans, Halomonas aquamarina,
Halomonas
axialensis, Halomonas meridiana, Halomonas olivaria, Halomonas songnenensis,
Halomonas
variabilis, Herbaspirillum chlorophenolicum, Herbaspirillum frisingense,
Herbaspirillum hiltneri,
Herbaspirillum huttiense subsp. putei, Herbaspirillum lusitanum, Herminiimonas
fonticola,
Hydrogenophaga intermedia, Hydrogenophaga pseudoflava, Klebsiella oxytoca,
Kosakonia
sacchari, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus
modestisalitolerans,
Lactobacillus plantarum subsp. argentoratensis, Lactobacillus xiangfangensis,
Lechevalieria
roselyniae, Lentzea albida, Lentzea califomiensis, Leuconostoc camosum,
Leuconostoc
citreum, Leuconostoc gelidum subsp. gasicomitatum, Leuconostoc mesenteroides
subsp.
suionicum, Luteimonas aestuarii, Lysobacter antibioticus, Lysobacter
koreensis, Lysobacter
oryzae, Magnetospirillum moscoviense, Marinomonas alcarazii, Marinomonas
primoryensis,
Massilia aurea, Massilia jejuensis, Massilia kyonggiensis, Massilia timonae,
Mesorhizobium
acaciae, Mesorhizobium qingshengii, Mesorhizobium shonense, Methylobacterium
haplocladii,
Methylobacterium platani, Methylobacterium pseudosasicola, Methylobacterium
zatmanii,
Microbacterium oxydan, Micromonospora chaiyaphumensis, Micromonospora chalcea,
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46
Micromonospora citrea, Micromonospora coxensis, Micromonospora echinofusca,
Micromonospora halophytica, Micromonospora kangleipakensis, Micromonospora
maritima,
Micromonospora nigra, Micromonospora purpureochromo gene,
Micromonospora
rhizosphaerae, Micromonospora saelicesensis, Micro virga subterranea, Micro
virga zambiensis,
Mycobacterium alvei, Mycobacterium avium subsp. silvaticum, Mycobacterium
colombiense,
Mycobacterium conceptionense, Mycobacterium conceptionense, Mycobacterium
farcino genes,
Mycobacterium fortuitum subsp. fortuitum, Mycobacterium goodii, Mycobacterium
insubricum,
Mycobacterium Ilatzerense, Mycobacterium neoaurum, Mycobacterium
neworleansense,
Mycobacterium obuense, Mycobacterium peregrinum, Mycobacterium saopaulense,
Mycobacterium septicum, Mycobacterium setense, Mycobacterium smegmatis,
Neisseria
sub flava, Nocardia lijiangensis, Nocardia thailandica, Novosphingobium
barchaimii,
Novosphingobium lindaniclasticum, Novosphingobium lindaniclasticum,
Novosphingobium
mathurense, Ochrobactrum pseudo grignonense, Oxalicibacterium solurbis,
Paraburkholderia
glathei, Paraburkholderia humi, Paraburkholderia phenazinium, Paraburkholderia
phytofirmans,
Paraburkholderia sordidicola, Paraburkholderia terricola, Paraburkholderia
xenovorans,
Paracoccus laeviglucosivorans, Patulibacter ginsengiterrae, Polymorphospora
rubra,
Porphyrobacter colymbi, Prevotella jejuni, Prevotella melaninogenica,
Propionibacterium acnes
subsp. elongatum, Proteus vulgaris, Pro videncia rustigianii,
Pseudoalteromonas agarivorans,
Pseudoalteromonas atlantica, Pseudoalteromonas paragorgicola, Pseudomonas
asplenii,
Pseudomonas asuensis, Pseudomonas benzenivorans, Pseudomonas cannabina,
Pseudomonas cissicola, Pseudomonas con gelans, Pseudomonas costantinii,
Pseudomonas
ficuserectae, Pseudomonas frederiksbergensis, Pseudomonas graminis,
Pseudomonas
jessenii, Pseudomonas koreensis, Pseudomonas koreensis, Pseudomonas
kunmingensis,
Pseudomonas marginalis, Pseudomonas mucidolens, Pseudomonas panacis,
Pseudomonas
plecoglossicida, Pseudomonas poae, Pseudomonas pseudoalcaligenes, Pseudomonas
putida,
Pseudomonas reinekei, Pseudomonas rhizosphaerae, Pseudomonas
seleniipraecipitans,
Pseudomonas umsongensis, Pseudomonas zhaodongensis, Pseudonocardia
alaniniphila,
Pseudonocardia ammonioxydans, Pseudonocardia autotrophica, Pseudonocardia
kongjuensis,
Pseudonocardia yunnanensis, Pseudorhodoferax soli, Pseudoxanthomonas
daejeonensis,
Pseudoxanthomonas indica, Pseudoxanthomonas kaohsiungensis, Psychrobacter
aquaticus,
Psychrobacter arcticus, Psychrobacter celer, Psychrobacter marincola,
Psychrobacter
nivimaris, Psychrobacter okhotskensis, Psychrobacter okhotskensis,
Psychrobacter piscatorii,
Psychrobacter pulmonis, Ramlibacter ginsenosidimutans, Rheinheimera japonica,
Rheinheimera muenzenbergensis, Rheinheimera soli, Rheinheimera tangshanensis,
Rheinheimera texasensis, Rheinheimera tilapiae, Rhizobium alamii, Rhizobium
azibense,
Rhizobium binae, Rhizobium daejeonense, Rhizobium endophyticum, Rhizobium
etli,
Rhizobium fabae, Rhizobium freirei, Rhizobium gallicum, Rhizobium loessense,
Rhizobium
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sophoriradicis, Rhizobium taibaishanense, Rhizobium mills, Rhizobium vignae,
Rhizobium
vignae, Rhizobium yanglingense, Rhodococcus baikonurensis, Rhodococcus
enclensis,
Rhodoferax saidenbachensis, Rickettsia canadensis, Rickettsia
heilongjiangensis, Rickettsia
honei, Rickettsia raoultii, Roseateles aquatilis, Roseateles aquatilis,
Salmonella enterica subsp.
salamae, Serratia ficaria, Serratia myotis, Serratia vespertilionis,
Shewanella aestuarii,
Shewanella decolorationis, Sphingobium amiense, Sphingobium baderi,
Sphingobium barthaii,
Sphingobium chlorophenolicum, Sphingobium cupriresistens, Sphingobium
czechense,
Sphingobium fuliginis, Sphingobium indicum, Sphingobium indicum, Sphingobium
japonicum,
Sphingobium lactosutens, Sphingomonas dokdonensis, Sphingomonas
pseudosanguinis,
Sphingopyxis chilensis, Sphingopyxis fribergensis, Sphingopyxis granuli,
Sphingopyxis indica,
Sphingopyxis witflariensis, Staphylococcus agnetis, Staphylococcus aureus
subsp. aureus,
Staphylococcus epidermidis, Staphylococcus hominis subsp. novobiosepticus,
Staphylococcus
nepalensis, Staphylococcus saprophyticus subsp. bovis, Staphylococcus sciuri
subsp.
camaticus, Streptomyces caeruleatus, Streptomyces canarius, Streptomyces
capoamus,
Streptomyces ciscaucasicus, Streptomyces griseorubiginosus, Streptomyces
olivaceoviridis,
Streptomyces panaciradicis, Streptomyces phaeopurpureus, Streptomyces pseudo
venezuelae,
Streptomyces resistomycificus, Tianweitania sediminis, Tsukamurella
paurometabola,
Variovorax guangxiensis, Vogesella alkaliphila, Xanthomonas arboricola,
Xanthomonas
axonopodis, Xanthomonas cassavae, Xanthomonas cucurbitae, Xanthomonas cynarae,
Xanthomonas euvesicatoria, Xanthomonas fragariae, Xanthomonas gardneri,
Xanthomonas
perforans, Xanthomonas pisi, Xanthomonas populi, Xanthomonas vasicola,
Xenophilus
aerolatus, Yersinia nurmii, Abiotrophia defectiva, Acidocella aminolytica,
Acinetobacter
guangdongensis, Acinetobacter parvus, Acinetobacter radioresistens,
Acinetobacter soli,
Acinetobacter variabilis, Actinomyces cardiffensis, Actinomyces dentalis,
Actinomyces
europaeus, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces
haliotis,
Actinomyces johnsonii, Actinomyces massiliensis, Actinomyces meyeri,
Actinomyces meyeri,
Actinomyces naeslundii, Actinomyces neuii subsp. anitratus, Actinomyces
odontolyticus,
Actinomyces oris, Actinomyces turicensis, Actinomycetospora corticicola,
Actinotignum schaalii,
Aerococcus christensenii, Aerococcus urinae, Aeromicrobium flavum,
Aeromicrobium
massiliense, Aeromicrobium tamlense, Aeromonas sharmana, Aggregatibacter
aphrophilus,
Aggregatibacter segnis, Agrococcus baldri, Albibacter methylovorans,
Alcaligenes faecalis
subsp. faecalis, Algoriphagus ratkowskyi, Alkalibacterium olivapovliticus,
Alkalibacterium
pelagium, Alkalibacterium pelagium, Alloprevotella rava, Alsobacter
metallidurans, Amaricoccus
kaplicensis, Amaricoccus veronensis, Anaerococcus hydrogenalis, Anaerococcus
lactolyticus,
Anaerococcus murdochii, Anaerococcus octavius, Anaerococcus prevotii,
Anaerococcus
vagina/is, Aquabacterium citratiphilum, Aquabacterium olei, Aquabacterium
olei, Aquabacterium
parvum, Aquincola tertiaricarbonis, Arcobacter venerupis, Arsenicicoccus
bolidensis,
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48
Arthrobacter russicus, Asticcacaulis excentricus, Atopobium deltae, Atopobium
parvulum,
Atopobium rimae, Atopobium vaginae, Aureimonas altamirensis, Aureimonas
rubiginis, Azospira
oryzae, Azospirillum oryzae, Bacillus circulans, Bacillus drentensis, Bacillus
fastidiosus, Bacillus
lehensis, Bacillus oceanisediminis, Bacillus rhizosphaerae, Bacteriovorax
stolpii, Bacteroides
coagulans, Bacteroides dorei, Bacteroides fragilis, Bacteroides ovatus,
Bacteroides stercoris,
Bacteroides uniformis, Bacteroides vulgatus, Bdellovibrio bacteriovorus,
Bdellovibrio exovorus,
Belnapia moabensis, Belnapia soli, Blautia hansenii, Blautia obeum, Blautia
wexlerae, Bosea
lathyri, Brachybacterium fresconis, Brachybacterium muris, Brevibacterium
ammoniilyticum,
Brevibacterium casei, Brevibacterium epidermidis, Brevibacterium iodinum,
Brevibacterium
luteolum, Brevibacterium paucivorans, Brevibacterium pityocampae,
Brevibacterium sanguinis,
Brevundimonas albigilva, Brevundimonas diminuta, Brevundimonas vancanneytii,
Caenimonas
terrae, Calidifontibacter indicus, Campylobacter concisus, Campylobacter
gracilis,
Campylobacter hominis, Campylobacter rectus, Campylobacter showae,
Campylobacter
ureolyticus, Capnocytophaga gin givalis, Capnocytophaga leadbetteri,
Capnocytophaga
ochracea, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium
valva rum,
Camobacterium divergens, Catonella morbi, Caulobacter henricii, Cavicella
subterranea,
Cellulomonas xylanilytica, Cellvibrio vulgaris, Chitinimonas taiwanensis,
Chryseobacterium
arachidis, Chryseobacterium daecheongense, Chryseobacterium formosense,
Chryseobacterium formosense, Chryseobacterium greenlandense, Chryseobacterium
indolo genes, Chryseobacterium piscium, Chryseobacterium rigui,
Chryseobacterium solani,
Chryseobacterium taklimakanense, Chryseobacterium ureilyticum,
Chryseobacterium
ureilyticum, Chryseobacterium zeae, Chryseomicrobium aureum, Cloacibacterium
haliotis,
Cloacibacterium normanense, Cloacibacterium normanense, Collinsella
aerofaciens,
Comamonas denitrificans, Comamonas terrigena, Corynebacterium accolens,
Corynebacterium
afermentans subsp. lipophilum, Corynebacterium ammonia genes, Corynebacterium
amycolatum, Corynebacterium aurimucosum, Corynebacterium aurimucosum,
Corynebacterium coyleae, Corynebacterium durum, Corynebacterium freiburgense,
Corynebacterium glaucum, Corynebacterium glyciniphilum, Corynebacterium
imitans,
Corynebacterium jeikeium, Corynebacterium jeikeium, Corynebacterium
kroppenstedtii,
Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium
mastitidis,
Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium
mucifaciens,
Corynebacterium mustelae, Corynebacterium mycetoides, Corynebacterium
pyruviciproducens,
Corynebacterium simulans, Corynebacterium singulare, Corynebacterium sputi,
Corynebacterium suicordis, Corynebacterium tube rculostearicum,
Corynebacterium
tuberculostearicum, Corynebacterium ureicelerivorans, Corynebacterium
variabile,
Couchioplanes caeruleus subsp. caeruleus, Cupriavidus metallidurans,
Curtobacterium
herbarum, Dechloromonas agitata, Deinococcus actinosclerus, Deinococcus
antarcticus,
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49
Deinococcus caeni, Deinococcus ficus, Deinococcus geothermalis, Deinococcus
radiodurans,
Deinococcus wulumuqiensis, Deinococcus xinjiangensis, Dermabacter hominis,
Dermabacter
vagina/is, Dermacoccus nishinomiyaensis, Desemzia incerta, Desertibacter
roseus, Dialister
invisus, Dialister micraerophilus, Dialister propionicifaciens, Dietzia aura
ntiaca, Dietzia
cercidiphylli, Dietzia timorensis, Dietzia timorensis, Dokdonella koreensis,
Dokdonella
koreensis, Dolosigranulum pigrum, Eikenella corrodens, Elizabethkingia
miricola, Elstera
litoralis, Empedobacter brevis, Enhydrobacter aerosaccus, Enterobacter
xiangfangensis,
Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus olivae, Erwinia
rhapontici,
Eubacterium eligens, Eubacterium infirmum, Eubacterium rectale, Eubacterium
saphenum,
Eubacterium sulci, Exiguobacterium mexicanum, Facklamia tabacinasalis,
Falsirhodobacter
halotolerans, Finegoldia magna, Flavobacterium cutihirudinis, Flavobacterium
lindanitolerans,
Flavobacterium resistens, Friedmanniella capsulata, Fusobacterium nucleatum
subsp.
polymorphum, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis,
Gemella
sanguinis, Gemmobacter aquaticus, Gemmobacter caeni, Gordonia jinhuaensis,
Gordonia
kroppenstedtii, Gordonia polyisoprenivorans, Gordonia polyisoprenivorans,
Granulicatella
adiacens, Granulicatella elegans, Haemophilus parainfluenzae, Haemophilus
sputorum,
Halomonas sulfidaeris, Herpetosiphon aura ntiacus, Hydrocarboniphaga effusa,
Idiomarina
mans, Janibacter anophelis, Janibacter hoylei, Janibacter indicus, Janibacter
limosus,
Janibacter melonis, Jeotgalicoccus halophilus, Jonquetella anthropi, Kaistia
geumhonensis,
Kingella denitrificans, Kingella oralis, Klebsiella oxytoca, Knoellia
aerolata, Knoellia locipacati,
Kocuria atrinae, Kocuria camiphila, Kocuria kristinae, Kocuria palustris,
Kocuria turfanensis,
Lachnoanaerobaculum saburreum, Lachnoanaerobaculum saburreum, Lactobacillus
crispatus,
Lactobacillus iners, Lactococcus lactis subsp. lactis, Lactococcus lactis
subsp. lactis,
Lactococcus piscium, Lapillicoccus jejuensis, Lautropia mirabilis, Legionella
beliardensis,
Leptotrichia buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii,
Leptotrichia
hongkongensis, Leptotrichia shahii, Leptotrichia trevisanii, Leptotrichia
wadei, Luteimonas
terricola, Lysinibacillus fusiformis, Lysobacter spongiicola, Lysobacter
xinjiangensis,
Macrococcus caseolyticus, Marmoricola pocheonensis, Marmoricola scoriae,
Massilia
alkalitolerans, Massilia alkalitolerans, Massilia aurea, Massilia plicata,
Massilia timonae,
Megamonas rupellensis, Meiothermus silvanus, Methylobacterium dankookense,
Methylobacterium goesingense, Methylobacterium goesingense, Methylobacterium
isbiliense,
Methylobacterium jeotgali, Methylobacterium oxalidis, Methylobacterium
platani,
Methylobacterium pseudosasicola, Methyloversatilis universalis, Microbacterium
foliorum,
Microbacterium hydrothermale, Microbacterium hydrothermale, Microbacterium
lacticum,
Microbacterium lacticum, Microbacterium laevaniformans, Microbacterium
paludicola,
Microbacterium petrolearium, Microbacterium phyllosphaerae, Microbacterium
resistens,
Micrococcus antarcticus, Micrococcus cohnii, Micrococcus flavus, Micrococcus
lylae,
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Micrococcus terreus, Microlunatus aurantiacus, Micropruina glycogenica, Micro
virga aerilata,
Micro virga aerilata, Micro virga subterranea, Micro virga vignae, Micro virga
zambiensis,
Micro virgula aerodenitrificans, Mogibacterium timidum, Moraxella atlantae,
Moraxella
catarrhalis, Morganella morganii subsp. morganii, Morganella psychrotolerans,
Murdochiella
asaccharolytica, Mycobacterium asiaticum, Mycobacterium chubuense,
Mycobacterium
crocinum, Mycobacterium gadium, Mycobacterium holsaticum, Mycobacterium
iranicum,
Mycobacterium longobardum, Mycobacterium neoaurum, Mycobacterium neoaurum,
Mycobacterium obuense, Negativicoccus succinicivorans, Neisseria
bacilliformis, Neisseria
oralis, Neisseria sicca, Neisseria subflava, Nesterenkonia lacusekhoensis,
Nesterenkonia
rhizosphaerae, Nevskia persephonica, Nevskia ramosa, Niabella yanshanensis,
Niveibacterium
umoris, Nocardia niwae, Nocardia thailandica, Nocardioides agariphilus,
Nocardioides dilutus,
Nocardioides ganghwensis, Nocardioides hwasunensis, Nocardioides nanhaiensis,
Nocardioides sediminis, Nosocomiicoccus ampullae, Noviherbaspirillum malthae,
Novosphingobium lindaniclasticum, Novosphingobium rosa, Ochrobactrum
rhizosphaerae,
Olsenella uli, Omithinimicrobium murale, Omithinimicrobium tianjinense,
Oryzobacter terrae,
Otto wia beijingensis, Paenalcaligenes suwonensis, Paenibacillus
agaridevorans, Paenibacillus
phoenicis, Paenibacillus xylanexedens, Paludibacterium yongneupense, Pantoea
cypripedii,
Parabacteroides distasonis, Paraburkholderia andropogonis, Paracoccus
alcaliphilus,
Paracoccus angustae, Paracoccus kocurii, Paracoccus laeviglucosivorans,
Paracoccus
sediminis, Paracoccus sphaerophysae, Paracoccus yeei, Parvimonas micra,
Parviterribacter
multiflagellatus, Patulibacter ginsengiterrae, Pedobacter aquatilis,
Pedobacter ginsengisoli,
Pedobacter xixiisoli, Peptococcus niger, Peptoniphilus coxii, Peptoniphilus
gorbachii,
Peptoniphilus harei, Peptoniphilus koenoeneniae, Peptoniphilus lacrimalis,
Peptostreptococcus
anaerobius, Peptostreptococcus stomatis, Phascolarctobacterium faecium,
Phenylobacterium
haematophilum, Phenylobacterium kunshanense, Pluralibacter gergoviae,
Polymorphobacter
multimanifer, Porphyromonas bennonis, Porphyromonas endodontalis,
Porphyromonas
gingivalis, Porphyromonas gingivicanis, Porphyromonas pasteri, Porphyromonas
pogonae,
Porphyromonas somerae, Povalibacter uvarum, Prevotella aurantiaca, Prevotella
baroniae,
Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella copri,
Prevotella corporis,
Prevotella denticola, Prevotella enoeca, Prevotella histicola, Prevotella
intermedia, Prevotella
jejuni, Prevotella jejuni, Prevotella maculosa, Prevotella melaninogenica,
Prevotella
melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella
nanceiensis, Prevotella
nigrescens, Prevotella oris, Prevotella oulorum, Prevotella pal/ens,
Prevotella pleuritidis,
Prevotella saccharolytica, Prevotella salivae, Prevotella shahii, Prevotella
timonensis, Prevotella
veroralis, Propionibacterium acidifaciens, Propionibacterium acnes subsp.
acnes,
Propionibacterium acnes subsp. acnes, Propionibacterium acnes subsp.
elongatum,
Propionibacterium granulosum, Propionimicrobium lymphophilum, Propionispira
arcuata,
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51
Pseudokineococcus lusitanus, Pseudomonas aeruginosa, Pseudomonas chengduensis,
Pseudonocardia benzenivorans, Pseudorhodoplanes sinuspersici, Psychrobacter
sanguinis,
Ramlibacter ginsenosidimutans, Rheinheimera aquimaris, Rhizobium alvei,
Rhizobium
daejeonense, Rhizobium larrymoorei, Rhizobium rhizoryzae, Rhizobium soli,
Rhizobium
taibaishanense, Rhizobium vignae, Rhodanobacter glycinis, Rhodobacter
veldkampii,
Rhodococcus enclensis, Rhodococcus fascians, Rhodococcus fascians, Rhodovarius
lipocyclicus, Rivicola pingtungensis, Roseburia inulinivorans, Rosenbergiella
nectarea,
Roseomonas aerilata, Roseomonas aquatica, Roseomonas mucosa, Roseomonas rosea,
Roseomonas vinacea, Rothia aeria, Rothia amarae, Rothia dentocariosa, Rothia
endophytica,
Rothia mucilaginosa, Rothia nasimurium, Rubellimicrobium mesophilum,
Rubellimicrobium
roseum, Rubrobacter bracarensis, Rudaea cellulosilytica, Ruminococcus gnavus,
Rune/la zeae,
Saccharopolyspora rectivirgula, Salinicoccus qingdaonensis, Scardo via
wiggsiae,
Sediminibacterium ginsengisoli, Selenomonas artemidis, Selenomonas infelix,
Selenomonas
noxia, Selenomonas sputigena, Shewanella aestuarii, Shuttleworthia satelles,
Simonsiella
muelleri, Skermanella aerolata, Skermanella stibiiresistens, Slackia exigua,
Smaragdicoccus
niigatensis, Sneathia sanguinegens, Solirubrobacter soli, Sphingobacterium
caeni,
Sphingobacterium daejeonense, Sphingobacterium hotanense, Sphingobacterium
kyonggiense, Sphingobacterium multivorum, Sphingobacterium nematocida,
Sphingobacterium
spiritivorum, Sphingobium amiense, Sphingobium indicum, Sphingobium
lactosutens,
Sphingobium subterraneum, Sphingomonas abaci, Sphingomonas aestuarii,
Sphingomonas
canadensis, Sphingomonas daechungensis, Sphingomonas dokdonensis, Sphingomonas
echinoides, Sphingomonas fonticola, Sphingomonas fonticola, Sphingomonas
formosensis,
Sphingomonas gei, Sphingomonas hankookensis, Sphingomonas hankookensis,
Sphingomonas koreensis, Sphingomonas kyeonggiensis, Sphingomonas laterariae,
Sphingomonas mucosissima, Sphingomonas oligophenolica, Sphingomonas
pseudosanguinis,
Sphingomonas sediminicola, Sphingomonas yantingensis, Sphingomonas
yunnanensis,
Sphingopyxis indica, Spirosoma rigui, Sporacetigenium mesophilum,
Sporocytophaga
myxococcoides, Staphylococcus auricularis, Staphylococcus epidermidis,
Staphylococcus
epidermidis, Staphylococcus hominis subsp. novobiosepticus, Staphylococcus
lugdunensis,
Staphylococcus pettenkoferi, Stenotrophomonas koreensis, Stenotrophomonas
rhizophila,
Stenotrophomonas rhizophila, Streptococcus agalactiae, Streptococcus canis,
Streptococcus
cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus
intermedius,
Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis,
Streptococcus
sanguinis, Streptomyces iconiensis, Streptomyces yanglinensis, Tabrizicola
aquatica,
Tahibacter caeni, Tannerella forsythia, Tepidicella xavieri, Tepidimonas
fonticaldi, Terracoccus
luteus, Tessaracoccus flavescens, Thermus thermophilus, Tianweitania
sediminis, Tianweitania
sediminis, Treponema amylovorum, Treponema denticola, Treponema
lecithinolyticum,
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Treponema medium, Turicella otitidis, Turicibacter sanguinis, Undibacterium
oligocarboniphilum, Undibacterium squillarum, Vagococcus salmoninarum,
Varibaculum
cambriense, Vibrio metschniko vii, Xanthobacter tagetidis, Xenophilus
aerolatus, Xenophilus
arseniciresistens, Yimella lutea, Zimmermannella alba, Zimmermannella bifida
or Zoo gloea
caeni.
[257] In other embodiments, the targeted bacteria cells are those commonly
found in the
vaginal microbiota and are, without limitation, Acinetobacter antiviralis,
Acinetobacter
baumannii, Acinetobacter calcoaceticus, Acinetobacter johnsonii, Actinobaculum
massiliense,
Actinobaculum schaalii, Actinomyces europaeus, Actinomyces graevenitzii,
Actinomyces
israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces neuii,
Actinomyces
odontolyticus, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces
viscosus,
Aerococcus christensenii, Aerococcus urinae, Aerococcus viridans, Aeromonas
encheleia,
Aeromonas salmonicida, Afipia massiliensis, Agrobacterium tumefaciens,
Algoriphagus
aquatilis, Aliivibrio wodanis, Alistipes finegoldii, Alloiococcus otitis,
Alloprevotella tannerae,
Alloscardovia omnicolens, Altererythrobacter epoxidivorans, Ammoniphilus
oxalaticus,
Amnibacterium kyonggiense, Anaerococcus hydrogenalis, Anaerococcus
lactolyticus,
Anaerococcus murdochii, Anaerococcus obesiensis, Anaerococcus prevotii,
Anaerococcus
tetradius, Anaerococcus vagina/is, Anaeroglobus geminatus, Anoxybacillus
pushchinoensis,
Aquabacterium parvum, Arcanobacterium phocae, Arthrobacter aurescens,
Asticcacaulis
excentricus, Atopobium minutum, Atopobium parvulum, Atopobium rimae, Atopobium
vaginae,
Avibacterium gallinarum, Bacillus acidicola, Bacillus atrophaeus, Bacillus
cereus, Bacillus cibi,
Bacillus coahuilensis, Bacillus gaemokensis, Bacillus methanolicus, Bacillus
oleronius, Bacillus
pumilus, Bacillus shackletonii, Bacillus sporothermodurans, Bacillus subtilis,
Bacillus wakoensis,
Bacillus weihenstephanensis, Bacteroides bamesiae, Bacteroides coagulans,
Bacteroides
dorei, Bacteroides faecis, Bacteroides forsythus, Bacteroides fragilis,
Bacteroides nordii,
Bacteroides ovatus, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides
uniformis,
Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides zoogleoformans,
Bamesiella
viscericola, Bhargavaea cecembensis, Bifidobacterium adolescentis,
Bifidobacterium bifidum,
Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium logum subsp.
infantis,
Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium
scardo vii,
Bilophila wadsworthia, Blautia hydrogenotrophica, Blautia obeum, Blautia
producta,
Brachybacterium faecium, Bradyrhizobium japonicum, Brevibacterium mcbrellneri,
Brevibacterium otitidis, Brevibacterium paucivorans, Bulleidia extructa,
Burkholderia fun gorum,
Burkholderia phenoliruptix, Caldicellulosiruptor saccharolyticus, Caldimonas
taiwanensis,
Campylobacter gracilis, Campylobacter hominis, Campylobacter sputorum,
Campylobacter
ureolyticus, Capnocytophaga ochracea, Cardiobacterium hominis, Catonella
morbi, Chlamydia
trachomatis, Chlamydophila abortus, Chondromyces robustus, Chryseobacterium
aquaticum,
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Citrobacter youngae, Cloacibacterium normanense, Clostridium cavendishii,
Clostridium
colicanis, Clostridium jejuense, Clostridium perfringens, Clostridium ramosum,
Clostridium
sordellii, Clostridium viride, Comamonas terrigena, Corynebacterium accolens,
Corynebacterium appendicis, Corynebacterium coyleae, Corynebacterium
glucuronolyticum,
Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium
kroppenstedtii,
Corynebacterium lipophiloflavum, Corynebacterium minutissimum, Corynebacterium
mucifaciens, Corynebacterium nuruki, Corynebacterium pseudo genitalium,
Corynebacterium
pyruviciproducens, Corynebacterium singulare, Corynebacterium striatum,
Corynebacterium
tuberculostearicum, Corynebacterium xerosis, Cryobacterium psychrophilum,
Curtobacterium
flaccumfaciens, Cutibacterium acnes, Cutibacterium avidum, Cytophaga
xylanolytica,
Deinococcus radiophilus, Delftia tsuruhatensis, Desulfovibrio desulfuricans,
Dialister invisus,
Dialister micraerophilus, Dialister pneumosintes, Dialister propionicifaciens,
Dickeya
chrysanthemi, Dorea longicatena, Eggerthella lenta, Eggerthia catenaformis,
Eikenella
corrodens, Enhydrobacter aerosaccus, Enterobacter asburiae, Enterobacter
cloacae,
Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus
faecium,
Enterococcus hirae, Erwinia persicina, Erwinia rhapontici, Erwinia toletana,
Escherichia coil,
Escherichia fergusonii, Eubacterium brachy, Eubacterium eligens, Eubacterium
nodatum,
Eubacterium rectale, Eubacterium saphenum, Eubacterium siraeum, Eubacterium
sulci,
Eubacterium yurii, Exiguobacterium acetylicum, Facklamia ignava,
Faecalibacterium prausnitzii,
Filifactor alocis, Finegoldia magna, Fusobacterium gonidiaformans,
Fusobacterium nucleatum,
Fusobacterium periodonticum, Gardnerella vagina/is, Gemella asaccharolytica,
Gemella
bergeri, Gemella haemolysans, Gemella sanguinis, Geobacillus
stearothermophilus,
Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Geobacter
grbiciae,
Granulicatella elegans, Haemophilus ducreyi, Haemophilus haemolyticus,
Haemophilus
parahaemolyticus, Haemophilus parainfluenzae, Hafnia alvei, Halomonas
meridiana,
Halomonas phoceae, Halomonas venusta, Herbaspirillum seropedicae,
Janthinobacterium
lividum, Jonquetella anthropi, Klebsiella granulomatis, Klebsiella oxytoca,
Klebsiella
pneumoniae, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus
brevis,
Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus curvatus,
Lactobacillus
delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus
helveticus,
Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii,
Lactobacillus kalixensis,
Lactobacillus kefiranofaciens, Lactobacillus kimchicus, Lactobacillus
kitasatonis, Lactobacillus
mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus
plantarum, Lactobacillus
pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus
salivarius, Lactobacillus
ultunensis, Lactobacillus vagina/is, Lactococcus lactis, Leptotrichia
buccalis, Leuconostoc
camosum, Leuconostoc citreum, Leuconostoc garlicum, Leuconostoc lactis,
Leuconostoc
mesenteroides, Lysinimonas kribbensis, Mageeibacillus indolicus, Maribacter
orientalis,
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Marinomonas protea, Marinospirillum insulare, Massilia timonae, Megasphaera
elsdenii,
Megasphaera micronuciformis, Mesorhizobium amorphae, Methylobacterium
radiotolerans,
Methylotenera versatilis, Microbacterium halophilum, Micrococcus luteus,
Microterricola viridarii,
Mobiluncus curtisii, Mobiluncus mu/lens, Mogibacterium timidum, Moore/la
glycerini, Moraxella
osloensis, Morganella morganii, Moryella indoligenes, Murdochiella
asaccharolytica,
Mycoplasma alvi, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma muris,
Mycoplasma salivarium, Negativicoccus succinicivorans, Neisseria flava,
Neisseria
gonorrhoeae, Neisseria mucosa, Neisseria sub flava, Nevskia ramosa, Nevskia
soli, Nitriliruptor
alkaliphilus, Odoribacter splanchnicus, Oligella urethra/is, Olsenella uli,
Paenibacillus
amylolyticus, Paenibacillus humicus, Paenibacillus pabuli, Paenibacillus
pasadenensis,
Paenibacillus pini, Paenibacillus validus, Pantoea agglomerans,
Parabacteroides merdae,
Paraburkholderia caryophylli, Paracoccus yeei, Parastreptomyces abscessus,
Parvimonas
micra, Pectobacterium betavasculorum, Pectobacterium carotovorum, Pediococcus
acidilactici,
Pediococcus ethanolidurans, Pedobacter alluvionis, Pedobacter wanjuense,
Pelomonas
aquatica, Peptococcus niger, Peptoniphilus asaccharolyticus, Peptoniphilus
gorbachii,
Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus lacrimalis,
Peptoniphilus
massiliensis, Peptostreptococcus anaerobius,
Peptostreptococcus massiliae,
Peptostreptococcus stomatis, Photobacterium angustum, Photobacterium
frigidiphilum,
Photobacterium phosphoreum, Porphyromonas asaccharolytica, Porphyromonas
bennonis,
Porphyromonas catoniae, Porphyromonas endodontalis, Porphyromonas gingivalis,
Porphyromonas somerae, Porphyromonas uenonis, Prevotella amnii, Prevotella
baroniae,
Prevotella bergensis, Prevotella bivia, Prevotella buccae, Prevotella
buccalis, Prevotella
colorans, Prevotella copri, Prevotella corporis, Prevotella dentalis,
Prevotella denticola,
Prevotella disiens, Prevotella intermedia, Prevotella loescheii, Prevotella
marshii, Prevotella
melaninogenica, Prevotella micans, Prevotella nigrescens, Prevotella oris,
Prevotella pleuritidis,
Prevotella ruminicola, Prevotella shahii, Prevotella stercorea, Prevotella
timonensis, Prevotella
veroralis, Propionimicrobium lymphophilum, Proteus mirabilis, Pseudomonas
abietaniphila,
Pseudomonas aeruginosa, Pseudomonas amygdali, Pseudomonas azotoformans,
Pseudomonas chlororaphis, Pseudomonas cuatrocienegasensis, Pseudomonas
fluorescens,
Pseudomonas fulva, Pseudomonas lutea, Pseudomonas mucidolens, Pseudomonas
oleovorans, Pseudomonas orientalis, Pseudomonas pseudoalcaligenes, Pseudomonas
psychrophila, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas syringae,
Pseudomonas tolaasii, Pseudopropionibacterium propionicum, Rahnella aquatilis,
Ralstonia
pickettii, Ralstonia solanacearum, Raoultella planticola, Rhizobacter dauci,
Rhizobium etli,
Rhodococcus fascians, Rhodopseudomonas palustris, Roseburia intestinalis,
Roseburia
inulinivorans, Rothia mucilaginosa, Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus torques, Sanguibacter keddieii, Sediminibacterium salmoneum,
Selenomonas
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bovis, Serratia fonticola, Serratia liquefaciens, Serratia marcescens,
Shewanella algae,
Shewanella amazonensis, Shigella boydii, Shigella sonnei, Slackia exigua,
Sneathia amnii,
Sneathia sanguinegens, Solobacterium moorei, Sorangium cellulosum, Sphingobium
amiense,
Sphingobium japonicum, Sphingobium yanoikuyae, Sphingomonas wittichii,
Sporosarcina
aquimarina, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus
capitis,
Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus
hominis,
Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus
schleiferi,
Staphylococcus simiae, Staphylococcus simulans, Staphylococcus wameri,
Stenotrophomonas
maltophilia, Stenoxybacter acetivorans, Streptococcus agalactiae,
Streptococcus anginosus,
Streptococcus australis, Streptococcus equinus, Streptococcus gallolyticus,
Streptococcus
infantis, Streptococcus intermedius, Streptococcus lutetiensis, Streptococcus
marimammalium,
Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus
parasanguinis,
Streptococcus phocae, Streptococcus pseudopneumoniae, Streptococcus saliva
rius,
Streptococcus sanguinis, Streptococcus thermophilus, Sutterella
wadsworthensis, Tannerella
forsythia, Terrahaemophilus aromaticivorans, Treponema denticola, Treponema
maltophilum,
Treponema parvum, Treponema vincentii, Trueperella bemardiae, Turicella
otitidis, Ureaplasma
parvum, Ureaplasma urealyticum, Varibaculum cambriense, Variovorax paradoxus,
Veil/one/la
atypica, Veil/one/la dispar, Veil/one/la montpelfierensis, Veil/one/la
parvula, Virgibacillus proomii,
Viridibacillus arenosi, Viridibacillus arvi, Weissella cibaria, Weissella
soli, Xanthomonas
campestris, Xanthomonas vesicatoria, Zobelfia laminariae or Zoogloea ramigera.
[258] In one embodiment, the targeted bacteria are Escherichia co/i.
[259] Thus, bacteriophages used for preparing the bacterial delivery vehicles,
and then the
bacterial delivery vehicles, may target (e.g., specifically target) a
bacterial cell from any one or
more of the foregoing genus and/or species of bacteria to specifically deliver
the payload of
interest.
[260] In one embodiment, the targeted bacteria are pathogenic bacteria. The
targeted bacteria
can be virulent bacteria.
[261] The targeted bacteria can be antibacterial resistance bacteria,
including those selected
from the group consisting of extended-spectrum beta-lactamase-producing (ESBL)
Escherichia
coil, ESBL Klebsiella pneumoniae, vancomycin-resistant Enterococcus (VRE),
meth icillin-
resistant Staphylococcus aureus (M RSA), multidrug-resistant (MDR)
Acinetobacter baumannii,
MDR Enterobacter spp., and a combination thereof. The targeted bacteria can be
selected from
the group consisting of extended-spectrum beta-lactamase-producing (ESBL)
Escherichia coli
strains. In a particular embodiment, said targeted bacteria are ESBL
Escherichia coli and/or
ESBL Klebsiella pneumoniae.
[262] Alternatively, the targeted bacterium can be a bacterium of the
microbiome of a given
species, including a bacterium of the human microbiota.
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[263] The present disclosure is directed to a bacterial delivery vehicle
containing the payload
as described herein. The bacterial delivery vehicles are typically prepared
from bacterial virus.
The bacterial delivery vehicles are typically chosen in order to be able to
introduce the payload
into the targeted bacteria.
[264] Bacterial viruses, from which the bacterial delivery vehicles disclosed
herein may be
derived, include bacteriophages. Optionally, the bacteriophage is selected
from the Order
Caudovirales consisting of, based on the taxonomy of Krupovic et al, Arch
Virol, 2015, the family
Myoviridae, the family Podoviridae, the family Siphoviridae, and the family
Ackermannviridae.
[265] Bacteriophages may be selected from the family Myoviridae (such as,
without limitation,
genus Cp220virus, Cp8virus, Ea214virus, Felixo1virus, Mooglevirus, Suspvirus,
Hp1virus,
P2virus, Kayvirus, P100virus, Silviavirus, Spo1virus, Tsarbombavirus,
Twortvirus, Cc31virus,
Jd18virus, Js98virus, Kp15virus, Moonvirus, Rb49virus, Rb69virus, S16virus,
5chizot4virus,
Sp18virus, T4virus, Cr3virus, Se1virus, V5virus, Abouovirus, Agatevirus,
Agrican357virus,
Ap22virus, Aryl virus, B4virus, Bastillevirus, Bc431virus, Bcep78virus,
Bcepmuvirus,
Biquartavirus, Bxz1virus, Cd119virus, Cp51virus, Cvm10virus, Eah2virus,
Elvirus, Hapunavirus,
Jimmervirus, Kpp1Ovirus, M12virus, Machinavirus, Marthavirus, Msw3virus,
Muvirus,
Myohalovirus, Nit1 virus, P1virus, Pakpunavirus, Pbunavirus, Phikzvirus,
Rheph4virus,
RsI2virus, Rslunavirus, 5ecunda5virus, Sep1virus, 5pn3viru5, Svunavirus,
Tg1virus, Vhmlvirus
and Wphvirus).
[266] Bacteriophages may be selected from the family Podoviridae (such as,
without limitation,
genus Fri1virus, Kp32virus, Kp34virus, Phikmvvirus, Pradovirus, 5p6viru5,
T7virus, Cp1virus,
P68virus, Phi29virus, Nona33virus, Pocjvirus, TI2011virus, Bcep22virus,
Bpp1virus,
Cba41virus, Df112virus, Ea92virus, Epsilon15virus, F116virus, G7cvirus,
Jwalphavirus, Kf1 virus,
Kpp25virus, Lit1 virus, Luz24virus, Luz7virus, N4virus, Nonanavirus, P22virus,
Pagevirus,
Phieco32virus, Prtbvirus, 5p58viru5, Una961virus and Vp5virus).
[267] Bacteriophages may be selected from the family Siphoviridae (such as,
without limitation,
genus Camvirus, Likavirus, R4virus, Acadianvirus, Coopervirus, Pg1virus,
Pipefishvirus,
Rosebushvirus, Brujitavirus, Che9cvirus, Hawkeyevirus, Plotvirus, Jerseyvirus,
K1gvirus,
Sp31virus, Lmd1virus, Una4virus, Bongovirus, Reyvirus, Buttersvirus,
Charlievirus, Redivirus,
Baxtervirus, Nymphadoravirus, Bignuzvirus, Fishburnevirus, Phayoncevirus,
Kp36virus,
Rogue1virus, Rtpvirus, Ti virus, Tlsvirus, Ab18virus, Amigovirus,
Anatolevirus, Andromedavirus,
Attisvirus, Barnyardvirus, Bernal13virus, Biseptimavirus, Bronvirus, C2virus,
C5virus,
Cba181virus, Cbastvirus, Cecivirus, Che8virus, Chivirus, Cjw1virus,
Corndogvirus, Cronusvirus,
D3112viru5, D3virus, Decurrovirus, Demosthenesvirus, Doucettevirus, E125virus,
Eiauvirus,
Ff47virus, Gaiavirus, Gilesvirus, Gordonvirus, Gordtnkvirus, Harrisonvirus,
Hk578virus,
Hk97virus, Jenstvirus, Jwxvirus, Kelleziovirus, Korravirus, L5virus,
lambdavirus, Laroyevirus,
Liefievirus, Marvinvirus, Mudcatvirus, N15virus, Nonagvirus, Np1virus,
Omegavirus,
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P12002virus, P12024virus, P23virus, P7Ovirus, Pa6virus, Pamx74virus,
Patiencevirus,
Pbi1virus, Pepy6virus, Pfr1virus, Phic31virus, Phicbkvirus, Phietavirus,
Phifelvirus, PhijI1virus,
Pis4avirus, Psavirus, Psimunavirus, Rdjlvirus, Rer2virus, Sap6virus,
Send513virus,
Septima3virus, Seuratvirus, Sextaecvirus, Sfi11virus, Sfi21dt1virus,
Sitaravirus, Sk1virus,
Slashvirus, Smoothievirus, Soupsvirus, Spbetavirus, Ssp2virus, T5virus,
Tankvirus, Tin2virus,
Titanvirus, Tm4virus, Tp21virus, Tp84virus, Triavirus, Trigintaduovirus,
Vegasvirus,
Vendettavirus, Wbetavirus, Wildcatvirus, Wizardvirus, Woesvirus, Xp1Ovirus,
Ydn12virus and
Yuavirus).
[268] Bacteriophages may be selected from the family Ackermannviridae (such
as, without
limitation, genus Ag3virus, Limestonevirus, Cba120virus and Vii virus).
[269] Optionally, the bacteriophage is not part of the order Caudovirales but
from families with
unassigned order such as, without limitation, family Tectiviridae (such as
genus Alphatectivirus,
Betatectivirus), family Corticoviridae (such as genus Corticovirus), family
Inoviridae (such as
genus Fibrovirus, Habenivirus, lnovirus, Lineavirus, Plectrovirus, Saetivirus,
Vespertiliovirus),
family Cystoviridae (such as genus Cystovirus), family Leviviridae (such as
genus Allolevivirus,
Levivirus), family Micro viridae (such as genus Alpha3microvirus,
G4microvirus,
Phix174microvirus, Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus) and
family
Plasmaviridae (such as genus Plasmavirus).
[270] Optionally, the bacteriophage is targeting Archea not part of the Order
Caudovirales but
from families with unassigned order such as, without limitation,
Ampullaviridae, FuselloViridae,
Globuloviridae, Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae,
Salterpro virus and
Bicaudaviridae.
[271] A non-exhaustive listing of bacterial genera and their known host-
specific bacteria
viruses is presented in the following paragraphs. The chimeric RBPs and/or the
recombinant
gpJ proteins and/or the recombinant gpH proteins, and the bacterial delivery
vehicles disclosed
herein may be engineered, as non-limiting examples, from the following phages.
Synonyms and
spelling variants are indicated in parentheses. Homonyms are repeated as often
as they occur
(e.g., D, D, d). Unnamed phages are indicated by "NN" beside their genus and
their numbers
are given in parentheses.
[272] Bacteria of the genus Actinomyces can be infected by the following
phages: Av-I, Av-2,
Av-3, BF307, CTI, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.
[273] Bacteria of the genus Aeromonas can be infected by the following phages:
AA-I, Aeh2,
N, PMI, TP446, 3,4, 11, 13, 29, 31, 32, 37, 43, 43-10T, Si, 54, 55R.1, 56,
56RR2, 57, 58, 59.1,
60, 63, Aehl, F, PM2, 1, 25, 31, 40RR2.8t, (syn= 44R), (syn= 44RR2.8t), 65,
PM3, PM4, PM5
and PM6.
[274] Bacteria of the genus Bacillus can be infected by the following phages:
A, aizl, Al-K-I, B,
BCJAI, BC!, BC2, BLLI, BLI, BP142, BSLI, BSL2, BSI, B53, B58, BS15, BS18,
B522, B526,
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BS28, B531, B5104, B5105, B5106, BIB, B1715V1, C, 0K-I, Coll, Carl, OP-53, CS-
1, CSi, D,
D, D, D5, entl, FP8, FP9, FSi, F52, F53, F55, F58, F59, G, GH8, GT8, GV-I, GV-
2, GT-4, g3,
gI2, g13, g14, gI6, gI7, g21, g23, g24, g29, H2, kenl, KK-88, Kuml, Kyul, J7W-
1, LP52, (syn= LP-
52), L7, Mexl, MJ-I, m0r2, MP-7, MPIO, MP12, MP14, MP15, Neal, N 2, N5, N6P,
PBC!, PBLA,
PBPI, P2, S-a, SF2, SF6, Shal, Sill, 5P02, (syn= (1)SPP1), SP[3, STI, STi, SU-
II, t, Tbl, Tb2, Tb5,
TbI0, Tb26, Tb51, Tb53, Tb55, Tb77, Tb97, Tb99, Tb560, Tb595, Td8, Td6, TdI5,
Tg I, Tg4, Tg6,
Tg7, Tg9, TgI0, Tgll, TgI3, TgI5, Tg21, Tinl, Tin7, Tin8, TinI3, Tm3, Tad,
Togl, toll, TP-1, TP-
10vir, TP-15c, TP-16c, TP-17c, TP-19, 1P35, 1P51, TP-84, Tt4, Tt6, type A,
type B, type C, type
D, type E, T(p3, VA-9, W, wx23, wx26, Yunl, a, y, pllõ (pmed-2, (pT, (pp-4,
(p3T, (p75, (p105, (syn=
(p105), IA, IB, 1-97A, 1-97B, 2, 2, 3, 3, 3, 5, 12, 14, 20, 30, 35, 36, 37,
38, 41C, 51, 63, 64, 138D,
I, II, IV, NN-Bacillus (13), alel, ARI, AR2, AR3, AR7, AR9, Bace-11, (syn=
11), Bastille, BLI, BL2,
BL3, BL4, BLS, BL6, BL8, BL9, BP124, B528, B580, Ch, OP-Si, CP-54, D-5, darl,
denl, DP-7,
entl, FoSi, FoS2, F54, F56, F57, G, gall, gamma, GEI, GF-2, GSi, GT-1, GT-2,
GT-3, GT-4, GT-
5, GT-6, GT-7, GV-6, gI5, 19, 110, ISi, K, MP9, MP13, MP21, MP23, MP24, MP28,
MP29, MP30,
MP32, MP34, MP36, MP37, MP39, MP40, MP41, MP43, MP44, MP45, MP47, MP50, NLP-I,
No.1, N17, N19, PBSI, PKI, PMBI, PMB12, PMJI, S, SPOI, 5P3, 5P5, 5P6, 5P7,
5P8, 5P9, SPIO,
SP-15, 5P50, (syn= SP-50), 5P82, SST, subl, SW, Tg8, TgI2, TgI3, TgI4, thul,
thuA, thuS, Tin4,
Tin23, TP-13, 1P33, IPSO, TSP-I, type V, type VI, V, Vx, 1322, (pe, (pNR2,
(p25, (p63, 1, 1, 2, 20,
3N1, 4, 5, 6, 7, 8, 9, 10, 12, 12, 17, 18, 19, 21, 138, III, 4 (B.
megateriwn), 4 (B. sphaericus),
AR13, BPP-I0, B532, BS107, B1, B2, GA-1, GP-I0, GV-3, GV-5, g8, MP20, MP27,
MP49, Nf,
PPS, PP6, SF5, TgI8, TP-I, Versailles, (pI5, (p29, 1-97, 837/IV, mI-Bacillus
(1), BatIO, BSLIO,
BSLI 1, B56, BSI I, BS16, B523, BS101, BS102, gI8, marl, PBLI, 5N45, thu2,
thu3, Tml, Tm2, TP-
20, TP21, TP52, type F, type G, type IV, HN-BacMus (3), BLE, (syn= Bc), B52,
B54, B55, B57,
BIO, B12, B520, BS21, F, MJ-4, PBA12, AP50, AP50-04, AP50-11, AP50-23, AP50-
26, AP50-
27 and Bam35. The following Bacillus-specific phages are defective: DLP10716,
DLP-11946,
DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW,
PBSX,
PBSY, PBSZ, phi, SPa, type 1 and p.
[275] Bacteria of the genus Bacteroides can be infected by the following
phages: ad 12, Baf-
44, Baf-48B, Baf-64, Bf-1, Bf-52, B40-8, F1, 131, (pAl, (pBrOl, (pBr02, 11,
67.1, 67.3, 68.1, mt-
Bacteroides (3), Bf42, Bf71, HN-Bdellovibrio (1) and BF-41.
[276] Bacteria of the genus Bordetella can be infected by the following
phages: 134 and NN-
Bordetella (3).
[277] Bacteria of the genus Borrelia can be infected by the following phages:
NN-Borrelia (1)
and NN-Borrelia (2).
[278] Bacteria of the genus Bruce/la can be infected by the following phages:
A422, Bk, (syn=
Berkeley), BM29, F0i, (syn= F01), (syn= FQI), D, FP2, (syn= FP2), (syn= FD2),
Fz, (syn=
Fz75/13), (syn= Firenze 75/13), (syn= Fi), Fi, (syn= F1), Fim, (syn= Flm),
(syn= Fim), FiU, (syn=
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FIU), (syn= FiU), F2, (syn= F2), F3, (syn= F3), F4, (syn= F4), F5, (syn= F5),
F6, F7, (syn= F7),
F25, (syn= F25), (syn= 25), F25U, (syn= F25u), (syn= F25U), (syn= F25V), F44,
(syn- F44),
F45, (syn= F45), F48, (syn= F48), 1, Im, M, MC/75, M51, (syn= M85), P, (syn=
D), 5708, R, Tb,
(syn= TB), (syn= Tbilisi), W, (syn= Wb), (syn= Weybridge), X, 3, 6, 7, 10/1,
(syn= 10), (syn= F8),
(syn= F8), 12m, 24/11, (syn= 24), (syn= F9), (syn= F9), 45/111, (syn= 45), 75,
84, 212/XV, (syn=
212), (syn= FiO), (syn= F10), 371/XXIX, (syn= 371), (syn= Fn), (syn= Fl 1) and
513.
[279] Bacteria of the genus Burkholderia can be infected by the following
phages: 0P75, NN-
Burkholderia (1) and 42.
[280] Bacteria of the genus Campylobacter can be infected by the following
phages: C type,
NTCC12669, NTCC12670, NTCC12671, NTCC12672, NTCC12673, NTCC12674,
NTCC12675, NTCC12676, NTCC12677, NTCC12678, NTCC12679, NTCC12680,
NTCC12681, NTCC12682, NTCC12683, NTCC12684, 32f, 111c, 191, NN-Campylobacter
(2),
Vfi-6, (syn= V19), VfV-3, V2, V3, V8, V16, (syn= Vfi-1), V19, V20(V45), V45,
(syn= V-45) and
NN-Campylobacter (1).
[281] Bacteria of the genus Chlamydia can be infected by the following phages:
Chpl.
[282] Bacteria of the genus Clostridium can be infected by the following
phages: CAKI, CAS,
Ca7, CE[3, (syn= 10), CEy, Cldl, c-n71, c-203 Tox-, DE[3, (syn= ID), (syn=
IDt0X+), HM3, KMI,
KT, Ms, NAI, (syn= Naltox+), PA1350e, Pf6, PL73, PL78, PL81, PI, P50, P5771,
P19402,
ICt0X+, 2Ct0X\ 2D3 (syn= 2Dt0X+), 30, (syn= 3Ctox+), 40, (syn= 4Ct0X+), 56,
111-1, NN-
Clostridium (61), NBIt0X+, al, CAI, HMT, HM2, PFI5 P-23, P-46, 0-05, Q-oe, 0-
16, 0-21, 0-26,
0-40, 0-46, S111, SA02, WA01, WA03, Wm, W523, 80, C, CA2, CA3, CPTI, CPT4, cl,
c4, c5,
HM7, H1 1/A1, H18/Ax, FW523, Hi58ZA1, K2ZA1, K21Z523, ML, NA2t0X; Pf2, Pf3,
Pf4, 59Z53,
541ZA1, 544Z523, a2, 41, 112Z523, 214/S23, 233/Ai, 234/S23, 235/S23, II-1, 11-
2, 11-3, NN-
Clostridium (12), CAI, F1, K, S2, 1, 5 and NN-Clostridium (8).
[283] Bacteria of the genus Corynebacterium can be infected by the following
phages: CGKI
(defective), A, A2, A3, A101, A128, A133, A137, A139, A155, A182, B, BF, B17,
B18, B51, B271,
B275, B276, B277, B279, B282, C, capi, CCI, CGI, CG2, 0G33, 0L31, Cog, (syn=
CGS), D, E,
F, H, H-I, hqi, hq2, 11ZH33, li/31, J, K, K, (syn= Ktox"), L, L, (syn= Ltox+),
M, MC-I, MC-2, MC-
3, MC-4, MLMa, N, 0, ovi, ov2, ov3, P, P, R, RP6, R529, S, T, U, UB1, ub2,
UHi, UH3, uh3,
uh5, uh6, 13, (syn= [3t0x+), 1311v64, 13vir, y, (syn= ytox-), yI9, 6, (syn=
O'ox+), p, (syn= ptox-), 1)9,
(p984, w, IA, 1/1180, 2, 2/1180, 5/1180, 5ad/9717, 7/4465, 8/4465, 8ad/10269,
10/9253,
13Z9253, 15/3148, 21/9253, 28, 29, 55, 2747, 2893, 4498 and 5848.
[284] Bacteria of the genus Enterococcus can be infected by the following
phages: DF78, F1,
F2, 1, 2,4, 14, 41, 867, DI, 5B24, 2BV, 182, 225, C2, C2F, E3, E62, D596, H24,
M35, P3, P9,
SBI01, S2, 2B11, 5, 182a, 705, 873, 881, 940, 1051, 1057, 21096C, NN-
Enterococcus (1), PEI,
F1, F3, F4, VD13, 1, 200, 235 and 341.
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[285] Bacteria of the genus Erysipelothrix can be infected by the following
phage: NN-
Eiysipelothrix (1).
[286] Bacteria of the genus Escherichia can be infected by the following
phages: BW73, B278,
D6, D108, E, El, E24, E41, F1-2, F1-4, F1-5, HI8A, Ff18B, i, MM, Mu, (syn=
mu), (syn= Mul), (syn=
Mu-1), (syn= MU-1), (syn= Mul), (syn= p), 025, Ph1-5, Pk, PSP3, PI, PID, P2,
P4 (defective), SI,
WT, TK13, TR73 (defective), TI, (p2, (p7, T92, ip (defective), 7 A, 8T, 9T, 15
(defective), 18, 28-
1, 186, 299, HH-Escherichia (2), AB48, CM, 04,016, DD-VI, (syn= Dd-Vi), (syn=
DDVI), (syn=
DDVi), E4, E7, E28, FII, F13, H, HI, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5,
ND6, ND-7, Ox-
I (syn= OXI), (syn= HF), Ox-2 (syn= 0x2), (syn= 0X2), Ox-3, Ox-4, Ox-5, (syn=
0X5), Ox-6, (syn=
66F), (syn= T66t), (syn= T66t-)5 0111, Ph1-1, RB42, RB43, RB49, RB69, S, Sal-
I, Sal-2, Sal-3,
Sal-4, Sal-5, Sal-6, 1023, 1045, Tull*-6, (syn= Tull*), TuIP-24, Tull*46, TuIP-
60, 12, (syn=
ganuTia), (syn= y), (syn= PC), (syn= P.C.), (syn= 1-2), (syn= 12), (syn= P4),
14, (syn= 1-4),
(syn= 14), 16, 135, al, 1, IA, 3, (syn= Ac3), 3A, 31+, (syn= 3), (syn= MI),
5T, (syn= T5), 92660,
CF0103, HK620, J, K, KIF, m59, no. A, no. E, no. 3, no. 9, N4, sd, (syn= Sd),
(syn= SD), (syn=
Sa)3 (syn= sd), (syn= SD), (syn= CD), 13, (syn= 1-3), (syn= 13), 17, (syn= 1-
7), (syn= 17),
WPK, W31, AH, TC3888, TK3, TK7, TK12, TV-1, 004-CF, 005, 006, 007, TI, TI.2,
T20, T95,
T263, T1092, TI, TII, (syn=TW), 08, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32,
38, 39, 42, 933W,
NN-Escherichia (1), Esc-7-11, AC30, CVX-5, Cl, DDUP, ECI, EC2, E21, E29, F1,
F265, F275,
Hi, HK022, HK97, (syn= (DHK97), HK139, HK253, HK256, K7, ND-I, no.D, PA-2, q,
S2, TI, (syn=
a), (syn= P28), (syn= 1-1), (syn= Tx), 130, 15, (syn= 1-5), (syn= 15), UC-I,
w, 134, y2, A (syn=
lambda), (syn= GA), cl)D326, Ty, 006, 1)7, 010, T80, x, (syn= Xi), (syn= TX),
(syn= Txi), 2, 4,
4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81,
AC95, HK243,
Kb, ZG/3A, 5, 5A, 21 EL, Hi 9-J and 933H.
[287] Bacteria of the genus Fusobacterium can be infected by the following
phages: NN-
Fusobacterium (2), fv83-554/3, fv88-531/2, 227, fv2377, fv2527 and fv8501.
[288] Bacteria of the genus Haemophilus can be infected by the following
phages: HPI, S2 and
N3.
[289] Bacteria of the genus Helicobacter can be infected by the following
phages: HPI and AA-
Helicobacter (1).
[290] Bacteria of the genus Klebsiella can be infected by the following
phages: A10-2, K14B,
K16B, K19, (syn= K19), K114, K115, K121, K128, K129, K132, K133, K135, KI106B,
KI171B, KI181B,
KI832B, A10-1, AO-1, A0-2, A0-3, FC3-10, K, KI1, (syn= KII), K12, (syn= K12),
K13, (syn= K13),
(syn= KI 70/11), K14, (syn= K14), K15, (syn= K15), K16, (syn= K16), K17, (syn=
K17), K18, (syn=
K18), K119, (syn= K19), K127, (syn= K127), K131, (syn= K131), K135, KI171B,
11, VI, IX, CI-1, K14B,
K18, K111, K112, K113, K116, K117, K118, K120, K122, K123, K124, K126, K130,
K134, KI106B, Kli65B,
KI328B, KLXI, K328, P5046, 11,380, III, IV, VII, VIII, FC3-11, K12B, (syn=
K12B), K125, (syn=
K125), KI42B, (syn= K142), (syn= K1 42B), KI181B, (syn= KII 81), (syn=
K1181B), K1765/1, (syn=
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K1765/1), KI842B, (syn= K1832B), KI937B, (syn= K1937B), LI, (p28, 7, 231, 483,
490, 632 and
864/100.
[291] Bacteria of the genus Lepitospira can be infected by the following
phages: LEI, LE3, LE4
and -NN-Leptospira (1).
[292] Bacteria of the genus Listeria can be infected by the following phages:
A511, 01761,
4211, 4286, (syn= B054), A005, A006, A020, A500, A502, A511, Al 18, A620,
A640, B012,
B021, B024, B025, B035, B051, B053, B054, B055, B056, B101, B110, B545, B604,
B653, 0707,
D441, HS047, HIOG, H8/73, H19, H21, H43, H46, H107, H108, HI 10, H163/84,
H312, H340,
H387, H391/73, H684/74, H924A, PSA, U153, TMLUP5, (syn= P35), 00241, 00611,
02971A,
029710, 5/476, 5/911, 5/939, 5/11302, 5/11605, 5/11704, 184, 575, 633,
699/694, 744, 900,
1090, 1317, 1444, 1652, 1806, 1807, 1921/959, 1921/11367, 1921/11500,
1921/11566,
1921/12460, 1921/12582, 1967, 2389, 2425, 2671, 2685, 3274, 3550, 3551, 3552,
4276, 4277,
4292, 4477, 5337, 5348/11363, 5348/11646, 5348/12430, 5348/12434, 10072,
113550,
11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 910716 and NN-
Listeria (15).
[293] Bacteria of the genus Morganella can be infected by the following phage:
47.
[294] Bacteria of the genus Mycobacterium can be infected by the following
phages: 13, AGI,
ALi, ATCC 11759, A2, B.03, BG2, BKI, BK5, butyricum, B-1, B5, B7, B30, B35,
Clark, Cl, 02,
DNAIII, DSP1, D4, D29, GS4E, (syn= GS4E), GS7, (syn= GS-7), (syn= GS7), IPa,
lacticola,
Legendre, Leo, L5, (syn= (1)L-5), MC-I, MC-3, MC-4, minetti, MTPHI 1, Mx4,
MyF3P/59a, phlei,
(syn= phlei 1), phlei 4, Polonus 11, rabinovitschi, smegmatis, TM4, TM9, TMIO,
TM20, Y7, Y10,
T630, IB, IF, IH, 1/1, 67, 106, 1430, B1, (syn= Bol), B24, D, D29, F-K, F-S,
HP, Polonus 1, Roy,
RI, (syn= RI-Myb), (syn= Ri), 11, 31, 40, 50, 103a, 103b, 128, 3111-D, 3215-D
and NN-
Mycobacterium (1).
[295] Bacteria of the genus Neisseria can be infected by the following phages:
Group!, group
!land NPI.
[296] Bacteria of the genus Nocardia can be infected by the following phages:
MNP8, NJ-L,
NS-8, N5 and TtiN-Nocardia.
[297] Bacteria of the genus Proteus can be infected by the following phages:
Pm5, 13vir, 2/44,
4/545, 6/1004, 13/807, 20/826, 57, 67b, 78, 107/69, 121, 9/0, 22/608, 30/680,
Pml, Pm3, Pm4,
Pm6, Pm7, Pm9, PmI0, Pmll, Pv2, 71, (p m, 7/549, 96/2, 10A/31, 12/55, 14, 15,
16/789, 17/971,
19A/653, 23/532, 25/909, 26/219, 27/953, 32A/909, 33/971, 34/13, 65, 5006M,
7480b, VI, 13/3a,
Clichy 12, 72600, cpx7, 1/1004, 5/742, 9, 12, 14, 22, 24/860, 2600/D52, Pm8
and 24/2514.
[298] Bacteria of the genus Pro videncia can be infected by the following
phages: PL25, PL26,
PL37, 9211/9295, 9213/921 lb, 9248, 7/R49, 7476/322, 7478/325, 7479, 7480,
9000/9402 and
9213/921 la.
[299] Bacteria of the genus Pseudomonas can be infected by the following
phages: Pf I, (syn=
Pf-I), Pf2, Pf3, PP7, PRRI, 7s, im-Pseudomonas (1), A1-1, A1-2, B 17, B89,
0B3, Col 2, Col 11,
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Col 18, Col 21, C154, C163, C167, C2121, E79, F8, ga, gb, H22, K1, M4, N2, Nu,
PB-1, (syn=
PBI), pfI6, PMN17, PPI, PP8, Psal, PsPI, PsP2, PsP3, PsP4, PsP5, PS3, P517,
PTB80, PX4,
PX7, PY0I, PY02, PY05, PY06, PY09, PY010, PY013, PY014, PY016, PY018, PY019,
PY020, PY029, PY032, PY033, PY035, PY036, PY037, PY038, PY039, PY041, PY042,
PY045, PY047, PY048, PY064, PY069, PY0103, PIK, SLPI, SL2, S2, UNL-I, wy, Yai,
Ya4,
Yan, TBE, TCTX, TC17, TKZ, (syn=c1)KZ), T-LT, Ornu78, TNZ, TPLS-1, TST-1, TW-
14, T-2,
1/72, 2/79, 3, 3/DO, 4/237, 5/406, 6C, 6/6660,7, 7v, 7/184, 8/280, 9/95,
10/502, 11/DE, 12/100,
125, 16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188, 337, 352, 1214, HN-
Pseudomonas (23),
A856, B26, CI-1, CI-2, C5, D, gh-1, Fl 16, HF, H90, K5, K6, KI 04, K109, K166,
K267, N4, N5,
06N-25P, PE69, Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8,
PP56,
PP87, PPI 14, PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy9220, psi,
PTB2, PTB20,
PTB42, PXI, PX3, PX10, PX12, PX14, PY070, PY071, R, SH6, 5H133, tf, Ya5, Ya7,
TBS,
cl)Kf77, T-MC, OrnnF82, TPLS27, TPLS743, TS-1, 1, 2, 2, 3, 4, 5, 6, 7, 7, 8,
9, 10, 11, 12, 12B,
13, 14,15, 14, 15, 16, 17, 18, 19, 20, 20, 21, 21, 22, 23, 23, 24, 25, 31, 53,
73, 119x, 145, 147,
170, 267, 284, 308, 525, NN-Pseudomonas (5), af, A7, B3, B33, B39, BI-1, C22,
D3, D37, D40,
D62, D3112, F7, F10, g, gd, ge, g HwI2, Jb 19, KFI, L , OXN-32P, 06N-52P, PCH-
I, PC13-1,
PC35-1, PH2, PH51, PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69,
PM105,
PMI 13, PM681, PM682, PO4, PPI, PP4, PPS, PP64, PP65, PP66, PP71, PP86, PP88,
PP92,
PP401, PP711, PP891, Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4,
P5-I0,
Pz, SDI, SLI, SL3, SL5, SM, TC5, TCI 1, TCII-1, TC13, TC15, TMO, TX, T04, T11,
(p240,2, 2F,
5, 7m, 11, 13, 13/441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218,
222, 236, 242, 246,
249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1, 400-1, HN-Pseudomonas
(6), G101,
M6, M6a, LI, PB2, PssyI5, Pssy4210, Pssy4220, PY012, PY034, PY049, PY050,
PY051,
PY052, PY053, PY057, PY059, PY0200, PX2, PX5, 5L4, T03, T06 and 1214.
[300] Bacteria of the genus Rickettsia can be infected by the following phage:
NN-Rickettsia.
[301] Bacteria of the genus Salmonella can be infected by the following
phages: b, Beccles,
CT, d, Dundee, f, Fels 2, GI, GUI, GVI, GVIII, k, K, i, j, L, 01, (syn= 0-1),
(syn= 01), (syn= 0-1),
(syn= 7), 02, 03, P3, P9a, PIO, 5ab3, 5ab5, SanIS, 5anI7, SI, Taunton, Vil,
(syn= Vil), 9,
imSalmonella (1), N-1, N-5, N-10, N-17, N-22, 11, 12, 16-19, 20.2, 36,
449C/C178, 966A/C259,
a, B.A.O.R., e, G4, GUI, L, LP7, M, MG40, N-18, P5A68, P4, P9c, P22, (syn=
P22), (syn=
PLT22), (syn= PLT22), P22al, P22-4, P22-7, P22-11, SNT-I, SNT-2, 5P6, Villi,
VilV, ViV, ViVI,
ViVII, Worksop, 5j5, c34, 1,37, 1(40), (syn= TI[40]), 1,422, 2, 2.5, 3b, 4, 5,
6,14(18), 8, 14(6,7),
10, 27, 28B, 30, 31, 32, 33, 34, 36, 37, 39, 1412, SNT-3, 7-11, 40.3, c, C236,
C557, C625,
C966N, g, GV, G5, GI 73, h, IRA, Jersey, MB78, P22-1, P22-3, P22-12, Sabl,
5ab2, 5ab2, 5ab4,
Sanl, 5an2, 5an3, 5an4, 5an6, 5an7, 5an8, 5an9, 5anI3, 5anI4, 5anI6, 5anI8,
5anI9, 5an20,
5an21, 5an22, 5an23, 5an24, 5an25, 5an26, SasLI, SasL2, SasL3, SasL4, SasL5,
SIBL, SII,
Vill, T1, 1,2, 3a, 3a1, 1010, Ym-Salmonella (1), N-4, SasL6 and 27.
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[302] Bacteria of the genus Serratia can be infected by the following phages:
A2P, P520,
SMB3, SMP, SMP5, SM2, V40, V56, ic, (PCP-3, (PCP-6, 3M, 10/1a, 20A, 34CC, 34H,
381, 345G,
345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228,
SLP,
SMPA, V.43, a, TCWI, cl)CP6-1, (1)CP6-2, (1)CP6-5, 31, 5, 8, 9F, 10/1, 20E,
32/6, 34B, 34C1,
34P, 37, 41, 56, 56D, 56P, 60P, 61/6, 74/6, 76/4, 101/8900, 226, 227, 228,
229F, 286, 289,
290F, 512, 764a, 2847/10, 2847/10a, L.359 and SMBI.
[303] Bacteria of the genus Shigella can be infected by the following phages:
Fsa, (syn=a),
FSD2d, (syn= D2d), (syn= W2d), FSD2E, (syn= W2e), fv, F6, f7.8, H-Sh, PE5,
P90, Sfll, Sh,
SHm, SHry, (syn= HIV), SHvi, (syn= HVI), SHVym, (syn= HVIII), SKy66, (syn=
gamma 66),
(syn= y1313), (syn= y66b), SKm, (syn= SIllb)5 (syn= UI), SKw, (syn= Siva),
(syn= IV), SICTM, (syn=
SIVA.), (syn= IVA), SKvi, (syn= KVI), (syn= Svi), (syn= VI), SKym, (syn= Svm),
(syn= VIII),
SKVI-11A, (syn= SymA), (syn= VIIIA), STvi, STK, STx1, STxn, S66, W2, (syn=
D2c), (syn= D20),
TI, TIVb 3-SO-R, 8368-SO-R, F7, (syn= F57), (syn= K29), F10, (syn= FS10),
(syn= K31), 11,
(syn= alfa), (syn= FSa), (syn= KI 8), (syn= a), 12, (syn= a), (syn= K19),
5G33, (syn= G35), (syn=
SO-35/G), 5G35, (syn= SO-55/G), 5G3201, (syn= SO-3201/G), SHn, (syn= HII),
SHy, (syn=
SHV), SHx, SHX, SKn, (syn= K2), (syn= KII), (syn= Sn), (syn= SsI1), (syn= II),
SKry, (syn= Sm),
(syn= SsIV), (syn= IV), SK1Va, (syn= Swab), (syn= SsIVa), (syn= IVa), SKV,
(syn= K4), (syn=
KV), (syn= SV), (syn= SsV), (syn= V), SKx, (syn= K9), (syn= KX), (syn= SX),
(syn= SsX), (syn=
X), STV, (syn= 135), (syn= 35-50-R), STym, (syn= 18345), (syn= 8345-SO-S-R),
W1, (syn= D8),
(syn= FSD8), W2a, (syn= D2A), (syn= FS2a), DD-2, Sf6, FSi, (syn= F1), SF6,
(syn= F6), 5G42,
(syn= SO-42/G), 5G3203, (syn= SO-3203/G), SKF12, (syn= SsF12), (syn= F12),
(syn= F12),
STn, (syn= 1881-SO-R), y66, (syn= gamma 66a), (syn= 55y66), (p2, BII, DDVII,
(syn= DD7),
FSD2b, (syn= W2B), F52, (syn= F2), (syn= F2), F54, (syn= F4), (syn= F4), F55,
(syn= F5),
(syn= F5), F59, (syn= F9), (syn= F9), Fl 1, P2-SO-S, 5G36, (syn= SO-36/G),
(syn= G36),
5G3204, (syn= SO-3204/G), 5G3244, (syn= SO-3244/G), SHi, (syn= HI), SHyrr,
(syn= HVII),
SHK, (syn= HIX), SHx1, SHx-rr, (syn= HXn), SKI, KI, (syn= Si), (syn= SsI),
SKVII, (syn= KVII),
(syn= Sv-rr), (syn= SsVII), SKIX, (syn= KIX), (syn= Six), (syn= SsIX), SKXII,
(syn= KXII), (syn=
Sxn), (syn= SsXII), STi, STffl, SIR', STVi, STyrr, S70, S206, U2-S0-S, 3210-SO-
S, 3859-SO-S,
4020-SO-S, (p3, (p5, (p7, (p8, (p9, TIO, (pl 1, (p13, (p14, (p18, SHm, (syn=
Hui), SHxi, (syn= HXt) and
SKxl, (syn= KXI), (syn= Sxi), (syn= SsXI), (syn= XI).
[304] Bacteria of the genus Staphylococcus can be infected by the following
phages: A, EW,
K, Ph5, Ph9, PhI0, PhI3, PI, P2, P3, P4, P8, P9, PIO, RG, SB-i, (syn= Sb-I),
S3K, Twort,
(1)SK311, (p812, 06, 40, 58, 119, 130, 131, 200, 1623, STCI, (syn=stc1), STC2,
(syn=5tc2),
44AHJD, 68, ACI, AC2, A6"C", A9"C", b581, CA-I, CA-2, CA-3, CA-4, CA-5, DI I,
L39x35, L54a,
M42, NI, N2, N3, N4, N5, N7, N8, NIO, Ni 1, N12, N13, N14, N16, Ph6, PhI2,
PhI4, UC-18, U4,
U15, SI, S2, S3, S4, S5, X2, Z1, TB5-2, TD, w, 11, (syn= (pl 1), (syn= P11-
M15), 15, 28, 28A, 29,
31, 31B, 37, 42D, (syn= P42D), 44A, 48, 51, 52, 52A, (syn= P52A), 52B, 53, 55,
69, 71, (syn=
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P71), 71A, 72, 75, 76, 77, 79, 80, 80a, 82, 82A, 83 A, 84, 85, 86, 88, 88A,
89, 90, 92, 95, 96,
102, 107, 108, 111, 129-26, 130, 130A, 155, 157, 157A, 165, 187, 275, 275A,
275B, 356, 456,
459, 471,471A, 489, 581, 676, 898, 1139, 1154A, 1259, 1314, 1380, 1405, 1563,
2148, 2638A,
2638B, 26380, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100, AC3,
A8, A10,
A13, b594n, D, HK2, N9, N15, P52, P87, SI, S6, Z4, TRE, 3A, 3B, 30, 6,7, 16,
21, 42B, 420,
42E, 44, 47, 47A5 470, 51, 54, 54x1, 70, 73, 75, 78, 81, 82, 88, 93, 94, 101,
105, 110, 115,
129/16, 174, 594n, 1363/14, 2460 and mS-Staphylococcus (1).
[305] Bacteria of the genus Streptococcus can be infected by the following
phages: EJ-I, NN-
Streptococais (1), a, Cl, FLOThs, H39, Op-I, Cp-5, Cp-7, Cp-9, Op-I0, A1298,
AS, alO/J1, alO/J2,
alO/J5, alO/J9, A25, BTII, b6, CAI, c20-1, c20-2, DP-I, Dp-4, DTI, E142, el0,
FA101, FEThs, FK,
FKKI01, FKLIO, FKP74, FKH, FLOThs, Fy101, fl, F10, F20140/76, g, GT-234, HB3,
(syn= HB-
3), HB-623, HB-746, M102, 01205, T01205, PST, PO, PI, P2, P3, P5, P6, P8, P9,
P9, P12,
P13, P14, P49, P50, P51, P52, P53, P54, P55, P56, P57, P58, P59, P64, P67,
P69, P71, P73,
P75, P76, P77, P82, P83, P88, sc, sch, sf, Sfll 1, (syn= SFil 1), (syn=
TSFill), (syn= (1)Sfill), (syn=
TSfill), sfil9, (syn= SFil9), (syn= TSFil9), (syn= TSfil9), Sfi21, (syn=
SFi21), (syn= TSFi21), (syn=
(pSfi21), STO, SIX, 5t2, ST2, ST4, S3, (syn= TS3), s265, 017, (p42, 057, (p80,
T81, (p82, (p83,
(p84, (p85, (p86, (p87, (p88, (p89, (p90, T91, (p92, (p93, (p94, (p95, (p96,
(p97, (p98, (p99, T100, T101,
(p102, (p227, 07201, wl, w2, w3, w4, w5, w6, w8, wI0, 1, 6, 9, 10F, 12/12, 14,
17SR, 19S, 24,
50/33, 50/34, 55/14, 55/15, 70/35, 70/36, 71/ST15, 71/45, 71/46, 74F, 79/37,
79/38, 80/J4,
80/J9, 80/ST16, 80/15, 80/47, 80/48, 101, 103/39, 103/40, 121/41, 121/42,
123/43, 123/44,
124/44, 337/ST17 and mStreptococcus (34).
[306] Bacteria of the genus Treponema can be infected by the following phage:
NN-
Treponema (1).
[307] Bacteria of the genus Vibrio can be infected by the following phages:
CTX(I), fs, (syn=
si), fs2, Ivpf5, VfI2, Vf33, VPI(1), VSK, v6, 493, OP-TI, E125, kappa, K139,
Labol, )XN-69P, OXN-
86, 06N-21P, PB-I, P147, rp-1, 5E3, VA-I, (syn= VcA-I), VcA-2, VPI, VP2, VP4,
VP7, VP8, VP9,
VPIO, VP17, VP18, VP19, X29, (syn= 29 d'Herelle), t, (1)HAW1-1, (1)HAW1-2,
(1)HAW1-3, (1)HAWI-
4, (1)HAW1-5, (1)HAW1-6, (1)HAW1-7, XHAWI-8, (1)HAW1-9, (1)HAW1-10, (1)HCI-1,
(1)HC1-2, (1)HC1-
3, (1)HC1-4, cl)HC2-1, >H02-2, cl)HC2-3, cl)HC2-4, (I)HC3-1, (I)HC3-2, (I)HC3-
3, (I)HD1S-1,
(1)HD1S-2, cl)HD2S-1, cl)HD2S-2, cl)HD2S-3, cl)HD2S-4, cl)HD2S-5, (1)HDO-1,
(1)HDO-2, cl)HDO-
3, (1)HDO-4, (1)HDO-5, (1)HDO-6, (I)KL-33, (I)KL-34, (1)KL-35, cl)KL-36,
(1)KWH-2, (1)KWH-3,
(1)KWH-4, cOMARQ-1, cOMARQ-2, cOMARQ-3, OMOAT-1, 1)0139, cOPEL1A-1, cOPEL1A-2,
cOPEL8A-1, cOPEL8A-2, cOPEL8A-3, cOPEL8C-1, cOPEL8C-2, cOPEL13A-1, cOPEL13B-1,
cOPEL13B-2, cOPEL13B-3, cOPEL13B-4, cOPEL13B-5, cOPEL13B-6, cOPEL13B-7,
cOPEL13B-8,
cOPEL13B-9, cOPEL13B-10, TVP143, TVP253, 016, (p138, 1-11, 5, 13, 14, 16, 24,
32, 493, 6214,
7050, 7227, 11, (syn= group 11), (syn== (p2), V, VIII, -m-Vibrio (13), KVP20,
KVP40, nt-1, 06N-
22P, P68, el, e2, e3, e4, e5, FK, G, 1, K, nt-6, NI, N2, N3, N4, N5, 06N-34P,
OXN-72P, OXN-
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4, 72A-10, 110A-
4, 333, 4996, I (syn= group l), Ill (syn= group III), VI, (syn= A-Saratov),
VII, IX, X, HN-Vibrio (6),
pAl, 7,7-8, 70A-2, 71A-6, 72A-5, 72A-8, 108A-10, 109A-6, 109A-8, I I0A-1, 110A-
5, 110A-7, hv-
1, OXN-52P, P13, P38, P53, P65, P108, Pill, 1PI3 VP3, VP6, VP12, VP13, 70A-3,
70A-4, 70A-
10, 72A-1, 108A-3, 109-61, 110A-2, 149, (syn= (p149), IV, (syn= group IV), NN-
Vibrio (22), VP5,
VPII, VP15, VP16, al, a2, a3a, a3b, 353B and HN-Vibrio (7).
[308] Bacteria of the genus Yersinia can be infected by the following phages:
H, H-I, H-2, H-3,
H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3/M64-76,
5/G394-76,
6/0753-76, 8/0239-76, 9/F18167, 1701, 1710, PST, 1/F2852-76, D'Herelle, EV, H,
Kotljarova,
PTB, R, Y, YerA41, TYer03-12, 3,4/01324-76, 7/F783-76, 903, 1/M6176 and
Yer2AT.
[309] In an embodiment, the bacteriophage is selected in the group consisting
of Salmonella
virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus
R02014, Escherichia
virus CBA120, Escherichia virus Phaxl, Salmonella virus 38, Salmonella virus
Det7, Salmonella
virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus
5H19,
Salmonella virus 5J3, Escherichia virus ECML4, Salmonella virus Marshall,
Salmonella virus
Maynard, Salmonella virus 5J2, Salmonella virus STML131, Salmonella virus Vil,
Erwinia virus
Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1,
Campylobacter virus 0P21, Campylobacter virus 0P220, Campylobacter virus
CPt10,
Campylobacter virus 16635, Campylobacter virus 0P81, Campylobacter virus
CP30A,
Campylobacter virus CPX, Campylobacter virus NCTC12673, Erwinia virus Ea214,
Erwinia virus
M7, Escherichia virus AY0145A, Escherichia virus E06, Escherichia virus HY02,
Escherichia
virus JH2, Escherichia virus TP1, Escherichia virus VpaE1, Escherichia virus
wV8, Salmonella
virus Felix01, Salmonella virus HB2014, Salmonella virus Mushroom, Salmonella
virus UAB87,
Citrobacter virus Moogle, Citrobacter virus Mordin, Escherichia virus SUSP1,
Escherichia virus
SUSP2, Aeromonas virus phi018P, Haemophilus virus HP1, Haemophilus virus HP2,
Pasteurella virus F108, Vibrio virus K139, Vibrio virus Kappa, Burkholderia
virus phi52237,
Burkholderia virus phiE122, Burkholderia virus phiE202, Escherichia virus 186,
Escherichia virus
P4, Escherichia virus P2, Escherichia virus Wphi, Mannheimia virus PHL101,
Pseudomonas
virus phiCTX, Ralstonia virus RSA1, Salmonella virus Fels2, Salmonella virus
PsP3, Salmonella
virus SopEphi, Yersinia virus L4130, Staphylococcus virus G1, Staphylococcus
virus G15,
Staphylococcus virus JD7, Staphylococcus virus K, Staphylococcus virus
M0E2014,
Staphylococcus virus P108, Staphylococcus virus Rodi, Staphylococcus virus
S253,
Staphylococcus virus S25-4, Staphylococcus virus 5Al2, Listeria virus A511,
Listeria virus
P100, Staphylococcus virus Remus, Staphylococcus virus SA11, Staphylococcus
virus 5tau2,
Bacillus virus Camphawk, Bacillus virus SP01, Bacillus virus B0P78, Bacillus
virus TsarBomba,
Staphylococcus virus Twort, Enterococcus virus phiEC24C, Lactobacillus virus
Lb338-1,
Lactobacillus virus LP65, Enterobacter virus PG7, Escherichia virus 0031,
Klebsiella virus
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JD18, Klebsiella virus PK0111, Escherichia virus Bp7, Escherichia virus IME08,
Escherichia
virus JS10, Escherichia virus JS98, Escherichia virus QL01, Escherichia virus
VR5,
Enterobacter virus Eap3, Klebsiella virus KP15, Klebsiella virus KP27,
Klebsiella virus Matisse,
Klebsiella virus Miro, Citrobacter virus Merlin, Citrobacter virus Moon,
Escherichia virus JSE,
Escherichia virus phi1, Escherichia virus RB49, Escherichia virus HX01,
Escherichia virus J509,
Escherichia virus RB69, Shigella virus UTAM, Salmonella virus S16, Salmonella
virus STML198,
Vibrio virus KVP40, Vibrio virus nt1, Vibrio virus VaIKK3, Escherichia virus
VR7, Escherichia
virus VR20, Escherichia virus VR25, Escherichia virus VR26, Shigella virus
SP18, Escherichia
virus AR1, Escherichia virus 040, Escherichia virus E112, Escherichia virus
E0ML134,
Escherichia virus HY01, Escherichia virus Ime09, Escherichia virus RB3,
Escherichia virus
RB14, Escherichia virus 14, Shigella virus Pss1, Shigella virus 5hfI2,
Yersinia virus D1, Yersinia
virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas virus Aeh1,
Escherichia
virus RB16, Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus
42,
Cronobacter virus CR3, Cronobacter virus CR8, Cronobacter virus CR9,
Cronobacter virus
PBES02, Pectobacterium virus phiTE, Cronobacter virus GAP31, Escherichia virus
4MG,
Salmonella virus SE1, Salmonella virus SSE121, Escherichia virus FFH2,
Escherichia virus FV3,
Escherichia virus JE52013, Escherichia virus V5, Brevibacillus virus Abouo,
Brevibacillus virus
Davies, Bacillus virus Agate, Bacillus virus Bobb, Bacillus virus Bp8pC,
Erwinia virus Deimos,
Erwinia virus Ea35-70, Erwinia virus RAY, Erwinia virus Simmy50, Erwinia virus
SpecialG,
Acinetobacter virus AB1, Acinetobacter virus AB2, Acinetobacter virus AbC62,
Acinetobacter
virus AP22, Arthrobacter virus ArV1, Arthrobacter virus Trina, Bacillus virus
AvesoBmore,
Bacillus virus B4, Bacillus virus Bigbertha, Bacillus virus Riley, Bacillus
virus Spock, Bacillus
virus Troll, Bacillus virus Bastille, Bacillus virus CAM003, Bacillus virus
Bc431, Bacillus virus
Bcp1, Bacillus virus BCP82, Bacillus virus BM15, Bacillus virus Deepblue,
Bacillus virus JBP901,
Burkholderia virus Bcep1, Burkholderia virus Bcep43, Burkholderia virus
Bcep781, Burkholderia
virus BcepNY3, Xanthomonas virus 0P2, Burkholderia virus BcepMu, Burkholderia
virus
phiE255, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacterium virus
Bxz1,
Mycobacterium virus Dandelion, Mycobacterium virus HyRo, Mycobacterium virus
13,
Mycobacterium virus Nappy, Mycobacterium virus Sebata, Clostridium virus
phiC2, Clostridium
virus phiCD27, Clostridium virus phiCD119, Bacillus virus CP51, Bacillus virus
JL, Bacillus virus
Shanette, Escherichia virus CVM10, Escherichia virus ep3, Erwinia virus
Asesino, Erwinia virus
EaH2, Pseudomonas virus EL, Halomonas virus HAP1, Vibrio virus VP882,
Brevibacillus virus
Jimmer, Brevibacillus virus Osiris, Pseudomonas virus Ab03, Pseudomonas virus
KPP10,
Pseudomonas virus PAKP3, Sinorhizobium virus M7, Sinorhizobium virus M12,
Sinorhizobium
virus N3, Erwinia virus Machina, Arthrobacter virus Brent, Arthrobacter virus
Jawnski,
Arthrobacter virus Martha, Arthrobacter virus Sonny, Edwardsiella virus MSW3,
Edwardsiella
virus PEi21, Escherichia virus Mu, Shigella virus SfMu, Halobacterium virus
phiH, Bacillus virus
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Grass, Bacillus virus NIT, Bacillus virus SPG24, Aeromonas virus 43,
Escherichia virus P1,
Pseudomonas virus CAb1, Pseudomonas virus CAb02, Pseudomonas virus JG004,
Pseudomonas virus PAKP1, Pseudomonas virus PAKP4, Pseudomonas virus PaP1,
Burkholderia virus BcepF1, Pseudomonas virus 141, Pseudomonas virus Ab28,
Pseudomonas
virus DL60, Pseudomonas virus DL68, Pseudomonas virus F8, Pseudomonas virus
JG024,
Pseudomonas virus KPP12, Pseudomonas virus LBL3, Pseudomonas virus LMA2,
Pseudomonas virus PB1, Pseudomonas virus SN, Pseudomonas virus PA7,
Pseudomonas
virus phiKZ, Rhizobium virus RHEph4, Ralstonia virus RSF1, Ralstonia virus
RSL2, Ralstonia
virus RSL1, Aeromonas virus 25, Aeromonas virus 31, Aeromonas virus Aes12,
Aeromonas
virus Aes508, Aeromonas virus A54, Stenotrophomonas virus IME13,
Staphylococcus virus
IPLAC1C, Staphylococcus virus SEP1, Salmonella virus SPN3US, Bacillus virus 1,
Geobacillus
virus GBSV1, Yersinia virus R1RT, Yersinia virus TG1, Bacillus virus G,
Bacillus virus PBS1,
Microcystis virus Ma-LMM01, Vibrio virus MAR, Vibrio virus VHML, Vibrio virus
VP585, Bacillus
virus BPS13, Bacillus virus Hakuna, Bacillus virus Megatron, Bacillus virus
WPh, Acinetobacter
virus AB3, Acinetobacter virus Abp1, Acinetobacter virus Fri1, Acinetobacter
virus IME200,
Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobacter virus
phiAB1,
Escherichia virus K30, Klebsiella virus K5, Klebsiella virus K11, Klebsiella
virus Kp1, Klebsiella
virus KP32, Klebsiella virus KpV289, Klebsiella virus F19, Klebsiella virus
K244, Klebsiella virus
Kp2, Klebsiella virus KP34, Klebsiella virus KpV41, Klebsiella virus KpV71,
Klebsiella virus
KpV475, Klebsiella virus 5U503, Klebsiella virus SU552A, Pantoea virus
Limelight, Pantoea
virus Limezero, Pseudomonas virus LKA1, Pseudomonas virus phiKMV, Xanthomonas
virus
f20, Xanthomonas virus f30, Xylella virus Prado, Erwinia virus Era103,
Escherichia virus K5,
Escherichia virus K1-5, Escherichia virus K1E, Salmonella virus 5P6,
Escherichia virus 17,
Kluyvera virus Kvp1, Pseudomonas virus gh1, Prochlorococcus virus PSSP7,
Synechococcus
virus P60, Synechococcus virus Syn5, Streptococcus virus Cp1, Streptococcus
virus Cp7,
Staphylococcus virus 44AHJD, Streptococcus virus C1, Bacillus virus B103,
Bacillus virus GA1,
Bacillus virus phi29, Kurthia virus 6, Actinomyces virus Av1, Mycoplasma virus
P1, Escherichia
virus 24B, Escherichia virus 933W, Escherichia virus Min27, Escherichia virus
PA28,
Escherichia virus 5tx2 11, Shigella virus 75025tx, Shigella virus P00J13,
Escherichia virus 191,
Escherichia virus PA2, Escherichia virus 1L2011, Shigella virus VASD,
Burkholderia virus
Bcep22, Burkholderia virus Bcepi102, Burkholderia virus Bcepmigl, Burkholderia
virus DC1,
Bordetella virus BPP1, Burkholderia virus BcepC6B, Cellulophaga virus Cba41,
Cellulophaga
virus Cba172, Dinoroseobacter virus DFL12, Erwinia virus Ea9-2, Erwinia virus
Frozen,
Escherichia virus phiV10, Salmonella virus Epsilon15, Salmonella virus SPN1S,
Pseudomonas
virus F116, Pseudomonas virus H66, Escherichia virus APEC5, Escherichia virus
APEC7,
Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virus ECBP1,
Escherichia virus
G7C, Escherichia virus IME11, Shigella virus Sb1, Achromobacter virus Axp3,
Achromobacter
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virus JWAlpha, Edwardsiella virus KF1, Pseudomonas virus KPP25, Pseudomonas
virus R18,
Pseudomonas virus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26,
Pseudomonas
virus Ab22, Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonas virus
PAA2,
Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonas virus TL,
Pseudomonas
virus KPP21, Pseudomonas virus LUZ7, Escherichia virus N4, Salmonella virus
9NA,
Salmonella virus 5P069, Salmonella virus BTP1, Salmonella virus HK620,
Salmonella virus P22,
Salmonella virus ST64T, Shigella virus Sf6, Bacillus virus Page, Bacillus
virus Palmer, Bacillus
virus Pascal, Bacillus virus Pony, Bacillus virus Pookie, Escherichia virus
172-1, Escherichia
virus ECB2, Escherichia virus NJ01, Escherichia virus phiEco32, Escherichia
virus Septima11,
Escherichia virus SU10, BruceIla virus Pr, BruceIla virus Tb, Escherichia
virus Pollock,
Salmonella virus FSL SP-058, Salmonella virus FSL SP-076, Helicobacter virus
1961P,
Helicobacter virus KHP30, Helicobacter virus KHP40, Hamiltonella virus APSE1,
Lactococcus
virus KSY1, Phormidium virus WMP3, Phormidium virus WMP4, Pseudomonas virus
119X,
Roseobacter virus SI01, Vibrio virus VpV262, Vibrio virus VC8, Vibrio virus
VP2, Vibrio virus
VP5, Streptomyces virus Amela, Streptomyces virus phiCAM, Streptomyces virus
Aaronocolus,
Streptomyces virus Caliburn, Streptomyces virus Danzina, Streptomyces virus
Hydra,
Streptomyces virus lzzy, Streptomyces virus Lannister, Streptomyces virus
Lika, Streptomyces
virus Sujidade, Streptomyces virus Zemlya, Streptomyces virus ELB20,
Streptomyces virus R4,
Streptomyces virus phiHau3, Mycobacterium virus Acadian, Mycobacterium virus
Baee,
Mycobacterium virus Reprobate, Mycobacterium virus Adawi, Mycobacterium virus
Bane1,
Mycobacterium virus BrownCNA, Mycobacterium virus Chrisnmich, Mycobacterium
virus
Cooper, Mycobacterium virus JAMaL, Mycobacterium virus Nigel, Mycobacterium
virus Stinger,
Mycobacterium virus Vincenzo, Mycobacterium virus Zemanar, Mycobacterium virus
Apizium,
Mycobacterium virus Manad, Mycobacterium virus Oline, Mycobacterium virus
Osmaximus,
Mycobacterium virus Pg1, Mycobacterium virus Soto, Mycobacterium virus
Suffolk,
Mycobacterium virus Athena, Mycobacterium virus Bernardo, Mycobacterium virus
Gadjet,
Mycobacterium virus Pipefish, Mycobacterium virus Godines, Mycobacterium virus
Rosebush,
Mycobacterium virus Babsiella, Mycobacterium virus Brujita, Mycobacterium
virus Che9c,
Mycobacterium virus Sbash, Mycobacterium virus Hawkeye, Mycobacterium virus
Plot,
Salmonella virus AG11, Salmonella virus Ent1, Salmonella virus f1 85E,
Salmonella virus Jersey,
Salmonella virus L13, Salmonella virus LSPA1, Salmonella virus 5E2, Salmonella
virus SETP3,
Salmonella virus SETP7, Salmonella virus SETP13, Salmonella virus SP101,
Salmonella virus
553e, Salmonella virus wksI3, Escherichia virus K1 G, Escherichia virus K1 H,
Escherichia virus
K1ind1, Escherichia virus K1ind2, Salmonella virus SP31, Leuconostoc virus
Lmd1,
Leuconostoc virus LN03, Leuconostoc virus LN04, Leuconostoc virus LN12,
Leuconostoc virus
LN6B, Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8,
Leuconostoc
virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34, Leuconostoc virus
LNTR3,
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Mycobacterium virus Bongo, Mycobacterium virus Rey, Mycobacterium virus
Butters,
Mycobacterium virus Michelle, Mycobacterium virus Charlie, Mycobacterium virus
Pipsqueaks,
Mycobacterium virus Xeno, Mycobacterium virus Panchino, Mycobacterium virus
Phrann,
Mycobacterium virus Redi, Mycobacterium virus Skinnyp, Gordonia virus
BaxterFox, Gordonia
virus Yeezy, Gordonia virus Kita, Gordonia virus Zirinka, Gorrdonia virus
Nymphadora,
Mycobacterium virus Bignuz, Mycobacterium virus Brusacoram, Mycobacterium
virus Donovan,
Mycobacterium virus Fishburne, Mycobacterium virus Jebeks, Mycobacterium virus
Malithi,
Mycobacterium virus Phayonce, Enterobacter virus F20, Klebsiella virus 1513,
Klebsiella virus
KLPN1, Klebsiella virus KP36, Klebsiella virus PKP126, Klebsiella virus Sushi,
Escherichia virus
AHP42, Escherichia virus AH524, Escherichia virus AK596, Escherichia virus
C119,
Escherichia virus E41c, Escherichia virus Eb49, Escherichia virus Jk06,
Escherichia virus KP26,
Escherichia virus Rogue1, Escherichia virus ACGM12, Escherichia virus Rtp,
Escherichia virus
ADB2, Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus Ti,
Shigella virus
PSf2, Shigella virus Shf11, Citrobacter virus Stevie, Escherichia virus TLS,
Salmonella virus
5P126, Cronobacter virus Esp2949-1, Pseudomonas virus Ab18, Pseudomonas virus
Ab19,
Pseudomonas virus PaMx11, Arthrobacter virus Amigo, Propionibacterium virus
Anatole,
Propionibacterium virus B3, Bacillus virus Andromeda, Bacillus virus Blastoid,
Bacillus virus
Curly, Bacillus virus Eoghan, Bacillus virus Finn, Bacillus virus Glittering,
Bacillus virus Riggi,
Bacillus virus Taylor, Gordonia virus Attis, Mycobacterium virus Barnyard,
Mycobacterium virus
Konstantine, Mycobacterium virus Predator, Mycobacterium virus Bernal13,
Staphylococcus
virus 13, Staphylococcus virus 77, Staphylococcus virus 108PVL, Mycobacterium
virus Bron,
Mycobacterium virus Faith1, Mycobacterium virus Joedirt, Mycobacterium virus
Rumpelstiltskin,
Lactococcus virus bIL67, Lactococcus virus c2, Lactobacillus virus c5,
Lactobacillus virus Ld3,
Lactobacillus virus Ld17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu,
Lactobacillus virus
phiLdb, Cellulophaga virus Cba121, Cellulophaga virus Cba171, Cellulophaga
virus Cba181,
Cellulophaga virus ST, Bacillus virus 250, Bacillus virus IEBH, Mycobacterium
virus Ardmore,
Mycobacterium virus Avani, Mycobacterium virus Boomer, Mycobacterium virus
Che8,
Mycobacterium virus Che9d, Mycobacterium virus Deadp, Mycobacterium virus
Diane,
Mycobacterium virus Dorothy, Mycobacterium virus Dotproduct, Mycobacterium
virus Drago,
Mycobacterium virus Fruitloop, Mycobacterium virus Gumbie, Mycobacterium virus
lbhubesi,
Mycobacterium virus Llij, Mycobacterium virus Mozy, Mycobacterium virus
Mutaforma13,
Mycobacterium virus Pacc40, Mycobacterium virus PMC, Mycobacterium virus
Ramsey,
Mycobacterium virus Rockyhorror, Mycobacterium virus 5G4, Mycobacterium virus
Shauna1,
Mycobacterium virus Shilan, Mycobacterium virus Spartacus, Mycobacterium virus
Taj,
Mycobacterium virus Tweety, Mycobacterium virus Wee, Mycobacterium virus
Yoshi,
Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088,
Salmonella
virus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244, Mycobacterium
virus Bask21,
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Mycobacterium virus CJW1, Mycobacterium virus Eureka, Mycobacterium virus
Kostya,
Mycobacterium virus Porky, Mycobacterium virus Pumpkin, Mycobacterium virus
Sirduracell,
Mycobacterium virus Toto, Mycobacterium virus Corndog, Mycobacterium virus
Firecracker,
Rhodobacter virus RcCronus, Pseudomonas virus D3112, Pseudomonas virus DMS3,
Pseudomonas virus FHA0480, Pseudomonas virus LPB1, Pseudomonas virus MP22,
Pseudomonas virus MP29, Pseudomonas virus MP38, Pseudomonas virus PA1KOR,
Pseudomonas virus D3, Pseudomonas virus PMG1, Arthrobacter virus Decurro,
Gordonia virus
Demosthenes, Gordonia virus Katyusha, Gordonia virus Kvothe, Propionibacterium
virus B22,
Propionibacterium virus Doucette, Propionibacterium virus E6,
Propionibacterium virus G4,
Burkholderia virus phi6442, Burkholderia virus phi1026b, Burkholderia virus
phiE125,
Edwardsiella virus eiAU, Mycobacterium virus Ff47, Mycobacterium virus Muddy,
Mycobacterium virus Gaia, Mycobacterium virus Giles, Arthrobacter virus
Captnmurica,
Arthrobacter virus Gordon, Gordonia virus GordTnk2, Paenibacillus virus
Harrison, Escherichia
virus EK99P1, Escherichia virus HK578, Escherichia virus JL1, Escherichia
virus SSL2009a,
Escherichia virus YD2008s, Shigella virus EP23, Sodalis virus 501, Escherichia
virus HK022,
Escherichia virus HK75, Escherichia virus HK97, Escherichia virus HK106,
Escherichia virus
HK446, Escherichia virus HK542, Escherichia virus HK544, Escherichia virus
HK633,
Escherichia virus mEp234, Escherichia virus mEp235, Escherichia virus mEpX1,
Escherichia
virus mEpX2, Escherichia virus mEp043, Escherichia virus mEp213, Escherichia
virus mEp237,
Escherichia virus mEp390, Escherichia virus mEp460, Escherichia virus mEp505,
Escherichia
virus mEp506, Brevibacillus virus Jenst, Achromobacter virus 83-24,
Achromobacter virus JWX,
Arthrobacter virus Kellezzio, Arthrobacter virus Kitkat, Arthrobacter virus
Bennie, Arthrobacter
virus DrRobert, Arthrobacter virus Glenn, Arthrobacter virus HunterDalle,
Arthrobacter virus
Joann, Arthrobacter virus Korra, Arthrobacter virus Preamble, Arthrobacter
virus Pumancara,
Arthrobacter virus Wayne, Mycobacterium virus Alma, Mycobacterium virus
Arturo,
Mycobacterium virus Astro, Mycobacterium virus Backyardigan, Mycobacterium
virus
BBPiebs31, Mycobacterium virus Benedict, Mycobacterium virus Bethlehem,
Mycobacterium
virus Bil!knuckles, Mycobacterium virus Bruns, Mycobacterium virus Bxb1,
Mycobacterium virus
Bxz2, Mycobacterium virus Che12, Mycobacterium virus Cuco, Mycobacterium virus
D29,
Mycobacterium virus Doom, Mycobacterium virus Ericb, Mycobacterium virus
Euphoria,
Mycobacterium virus George, Mycobacterium virus Gladiator, Mycobacterium virus
Goose,
Mycobacterium virus Hammer, Mycobacterium virus Heldan, Mycobacterium virus
Jasper,
Mycobacterium virus J027, Mycobacterium virus Jeffabunny, Mycobacterium virus
JHC117,
Mycobacterium virus KBG, Mycobacterium virus Kssjeb, Mycobacterium virus
Kugel,
Mycobacterium virus L5, Mycobacterium virus Lesedi, Mycobacterium virus
LHTSCC,
Mycobacterium virus lockley, Mycobacterium virus Marcell, Mycobacterium virus
Microwolf,
Mycobacterium virus Mrgordo, Mycobacterium virus Museum, Mycobacterium virus
Nepal,
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Mycobacterium virus Packman, Mycobacterium virus Peaches, Mycobacterium virus
Perseus,
Mycobacterium virus Pukovnik, Mycobacterium virus Rebeuca, Mycobacterium virus
Redrock,
Mycobacterium virus Ridgecb, Mycobacterium virus Rockstar, Mycobacterium virus
Saintus,
Mycobacterium virus Skipole, Mycobacterium virus Solon, Mycobacterium virus
Switzer,
Mycobacterium virus SWU1, Mycobacterium virus Ta17a, Mycobacterium virus
Tiger,
Mycobacterium virus Timshel, Mycobacterium virus Trixie, Mycobacterium virus
Turbido,
Mycobacterium virus Twister, Mycobacterium virus U2, Mycobacterium virus
Violet,
Mycobacterium virus Wonder, Escherichia virus DE3, Escherichia virus HK629,
Escherichia
virus HK630, Escherichia virus lambda, Arthrobacter virus Laroye,
Mycobacterium virus Halo,
Mycobacterium virus Liefie, Mycobacterium virus Marvin, Mycobacterium virus
Mosmoris,
Arthrobacter virus Circum, Arthrobacter virus Mudcat, Escherichia virus N15,
Escherichia virus
9g, Escherichia virus JenK1, Escherichia virus JenP1, Escherichia virus JenP2,
Pseudomonas
virus NP1, Pseudomonas virus PaMx25, Mycobacterium virus Baka, Mycobacterium
virus
Courthouse, Mycobacterium virus Littlee, Mycobacterium virus Omega,
Mycobacterium virus
Optimus, Mycobacterium virus Thibault, Polaribacter virus P12002L,
Polaribacter virus
P12002S, Nonlabens virus P12024L, Nonlabens virus P12024S, Thermus virus P23-
45,
Thermus virus P74-26, Listeria virus LP26, Listeria virus LP37, Listeria virus
LP110, Listeria
virus LP114, Listeria virus P70, Propionibacterium virus ATCC29399BC,
Propionibacterium
virus ATCC29399BT, Propionibacterium virus Attacne, Propionibacterium virus
Keiki,
Propionibacterium virus Kubed, Propionibacterium virus Lauchelly,
Propionibacterium virus
MrAK, Propionibacterium virus Ouroboros, Propionibacterium virus P91,
Propionibacterium
virus P105, Propionibacterium virus P144, Propionibacterium virus P1001,
Propionibacterium
virus P1.1, Propionibacterium virus P100A, Propionibacterium virus P100D,
Propionibacterium
virus P101A, Propionibacterium virus P104A, Propionibacterium virus PA6,
Propionibacterium
virus Pacnes201215, Propionibacterium virus PAD20, Propionibacterium virus
PAS50,
Propionibacterium virus PHLOO9M11, Propionibacterium virus PHL025M00,
Propionibacterium
virus PHL037M02, Propionibacterium virus PHL041M10, Propionibacterium virus
PHL060L00,
Propionibacterium virus PHL067M01, Propionibacterium virus PHL070N00,
Propionibacterium
virus PHL071N05, Propionibacterium virus PHL082M03, Propionibacterium virus
PHL092M00,
Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01,
Propionibacterium
virus PHL112N00, Propionibacterium virus PHL113M01, Propionibacterium virus
PHL114L00,
Propionibacterium virus PHL116M00, Propionibacterium virus PHL117M00,
Propionibacterium
virus PHL117M01, Propionibacterium virus PHL132N00, Propionibacterium virus
PHL141N00,
Propionibacterium virus PHL151M00, Propionibacterium virus PHL151N00,
Propionibacterium
virus PHL152M00, Propionibacterium virus PHL163M00, Propionibacterium virus
PHL171M01,
Propionibacterium virus PHL179M00, Propionibacterium virus PHL194M00,
Propionibacterium
virus PHL199M00, Propionibacterium virus PHL301M00, Propionibacterium virus
PHL308M00,
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Propionibacterium virus Pirate, Propionibacterium virus Procrass1,
Propionibacterium virus
SKKY, Propionibacterium virus Solid, Propionibacterium virus Stormborn,
Propionibacterium
virus Wizzo, Pseudomonas virus PaMx28, Pseudomonas virus PaMx74, Mycobacterium
virus
Patience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcus virus
Poco6,
Propionibacterium virus PFR1, Streptomyces virus phiBT1, Streptomyces virus
phiC31,
Streptomyces virus TG1, Caulobacter virus Karma, Caulobacter virus Magneto,
Caulobacter
virus phiCbK, Caulobacter virus Rogue, Caulobacter virus Swift, Staphylococcus
virus 11,
Staphylococcus virus 29, Staphylococcus virus 37, Staphylococcus virus 53,
Staphylococcus
virus 55, Staphylococcus virus 69, Staphylococcus virus 71, Staphylococcus
virus 80,
Staphylococcus virus 85, Staphylococcus virus 88, Staphylococcus virus 92,
Staphylococcus
virus 96, Staphylococcus virus 187, Staphylococcus virus 52a, Staphylococcus
virus 80a1pha,
Staphylococcus virus CNPH82, Staphylococcus virus EW, Staphylococcus virus
IPLA5,
Staphylococcus virus IPLA7, Staphylococcus virus IPLA88, Staphylococcus virus
PH15,
Staphylococcus virus phiETA, Staphylococcus virus phiETA2, Staphylococcus
virus phiETA3,
Staphylococcus virus phiMR11, Staphylococcus virus phiMR25, Staphylococcus
virus phiNM1,
Staphylococcus virus phiNM2, Staphylococcus virus phiNM4, Staphylococcus virus
5AP26,
Staphylococcus virus X2, Enterococcus virus FL1, Enterococcus virus FL2,
Enterococcus virus
FL3, Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcus
virus cIP1,
Aeromonas virus pIS4A, Listeria virus LP302, Listeria virus PSA,
Methanobacterium virus psiM1,
Roseobacter virus RDJL1, Roseobacter virus RDJL2, Rhodococcus virus RER2,
Enterococcus
virus BC611, Enterococcus virus IMEEF1, Enterococcus virus SAP6, Enterococcus
virus VD13,
Streptococcus virus SPQS1, Mycobacterium virus Papyrus, Mycobacterium virus
5end513,
Burkholderia virus KL1, Pseudomonas virus 73, Pseudomonas virus Ab26,
Pseudomonas virus
Kakheti25, Escherichia virus Cajan, Escherichia virus Seurat, Staphylococcus
virus SEP9,
Staphylococcus virus Sextaec, Streptococcus virus 858, Streptococcus virus
2972,
Streptococcus virus ALQ132, Streptococcus virus 01205, Streptococcus virus
ill,Sf
Streptococcus virus 7201, Streptococcus virus DT1, Streptococcus virus
phiAbc2,
Streptococcus virus 5fi19, Streptococcus virus 5fi21, Paenibacillus virus
Diva, Paenibacillus
virus Hb10c2, Paenibacillus virus Rani, Paenibacillus virus Shelly,
Paenibacillus virus Sitara,
Paenibacillus virus Willow, Lactococcus virus 712, Lactococcus virus A500191,
Lactococcus
virus A500273, Lactococcus virus A500281, Lactococcus virus A500465,
Lactococcus virus
A500532, Lactococcus virus Bibb29, Lactococcus virus bIL170, Lactococcus virus
0B13,
Lactococcus virus 0B14, Lactococcus virus 0B19, Lactococcus virus CB20,
Lactococcus virus
jj50, Lactococcus virus P2, Lactococcus virus P008, Lactococcus virus ski,
Lactococcus virus
SI4, Bacillus virus Slash, Bacillus virus Stahl, Bacillus virus Staley,
Bacillus virus Stills, Gordonia
virus Bachita, Gordonia virus ClubL, Gordonia virus OneUp, Gordonia virus
Smoothie, Gordonia
virus Soups, Bacillus virus SPbeta, Vibrio virus MAR10, Vibrio virus 55P002,
Escherichia virus
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AKFV33, Escherichia virus BF23, Escherichia virus DT57C, Escherichia virus
EPS7,
Escherichia virus FFH1, Escherichia virus H8, Escherichia virus 51ur09,
Escherichia virus 15,
Salmonella virus 1189705a12, Salmonella virus Shivani, Salmonella virus 5P035,
Salmonella
virus Stitch, Arthrobacter virus Tank, Tsukamurella virus 1IN2, Tsukamurella
virus 1IN3,
Tsukamurella virus 1IN4, Rhodobacter virus RcSpartan, Rhodobacter virus
RcTitan,
Mycobacterium virus Anaya, Mycobacterium virus Angelica, Mycobacterium virus
Crimd,
Mycobacterium virus Fionnbarth, Mycobacterium virus Jaws, Mycobacterium virus
Larva,
Mycobacterium virus Macncheese, Mycobacterium virus Pixie, Mycobacterium virus
TM4,
Bacillus virus BMBtp2, Bacillus virus TP21, Geobacillus virus Tp84,
Staphylococcus virus 47,
Staphylococcus virus 3a, Staphylococcus virus 42e, Staphylococcus virus
IPLA35,
Staphylococcus virus phi12, Staphylococcus virus phiSLT, Mycobacterium virus
32H0,
Rhodococcus virus RGL3, Paenibacillus virus Vegas, Gordonia virus Vendetta,
Bacillus virus
Wbeta, Mycobacterium virus Wildcat, Gordonia virus Twister6, Gordonia virus
Wizard, Gordonia
virus Hotorobo, Gordonia virus Monty, Gordonia virus Woes, Xanthomonas virus
CP1,
Xanthomonas virus OP1, Xanthomonas virus phi17, Xanthomonas virus Xop411,
Xanthomonas
virus Xp10, Streptomyces virus 1P1604, Streptomyces virus YDN12,
Alphaproteobacteria virus
phiJI001, Pseudomonas virus LK04, Pseudomonas virus M6, Pseudomonas virus
MP1412,
Pseudomonas virus PAE1, Pseudomonas virus Yua, Pseudoalteromonas virus PM2,
Pseudomonas virus phi6, Pseudomonas virus phi8, Pseudomonas virus phi12,
Pseudomonas
virus phi13, Pseudomonas virus phi2954, Pseudomonas virus phiNN, Pseudomonas
virus
phiYY, Vibrio virus fs1, Vibrio virus VGJ, Ralstonia virus R5603, Ralstonia
virus RSM1, Ralstonia
virus RSM3, Escherichia virus M13, Escherichia virus 122, Salmonella virus
IKe, Acholeplasma
virus L51, Vibrio virus fs2, Vibrio virus VFJ, Escherichia virus 1f1,
Propionibacterium virus B5,
Pseudomonas virus Pf1, Pseudomonas virus Pf3, Ralstonia virus PE226, Ralstonia
virus RSS1,
Spiroplasma virus SVTS2, Stenotrophomonas virus PSH1, Stenotrophomonas virus
SMA6,
Stenotrophomonas virus SMA7, Stenotrophomonas virus SMA9, Vibrio virus CTXphi,
Vibrio
virus KSF1, Vibrio virus VCY, Vibrio virus Vf33, Vibrio virus Vf03K6,
Xanthomonas virus Cf1c,
Spiroplasma virus 074, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x,
Escherichia
virus Fl, Escherichia virus Qbeta, Escherichia virus BZ13, Escherichia virus
M52, Escherichia
virus a1pha3, Escherichia virus 1D21, Escherichia virus 1D32, Escherichia
virus 1D62, Escherichia
virus N028, Escherichia virus N029, Escherichia virus N035, Escherichia virus
phiK,
Escherichia virus St1, Escherichia virus WA45, Escherichia virus G4,
Escherichia virus 1D52,
Escherichia virus Talmos, Escherichia virus phiX174, Bdellovibrio virus MAC1,
Bdellovibrio virus
MH2K, Chlamydia virus Chp1, Chlamydia virus Chp2, Chlamydia virus CPAR39,
Chlamydia
virus CPG1, Spiroplasma virus SpV4, Acholeplasma virus L2, Pseudomonas virus
PR4,
Pseudomonas virus PRD1, Bacillus virus AP50, Bacillus virus Bam35, Bacillus
virus GIL16,
Bacillus virus Wip1, Escherichia virus phi80, Escherichia virus RB42,
Escherichia virus 12,
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Escherichia virus 13, Escherichia virus 16, Escherichia virus VT2-Sa,
Escherichia virus VT1-
Sakai, Escherichia virus VT2-Sakai, Escherichia virus CP-933V, Escherichia
virus P27,
Escherichia virus 5tx2phi-1, Escherichia virus Stx1phi, Escherichia virus
5tx2phi-II, Escherichia
virus CP-1639õ based on the Escherichia virus BP-4795, Escherichia virus 86,
Escherichia
virus Min27, Escherichia virus 2851, Escherichia virus 1717, Escherichia virus
YYZ-2008,
Escherichia virus E0026 PO6, Escherichia virus EC0103 P15, Escherichia virus
EC0103 P12, Escherichia virus EC0111 P16, Escherichia virus EC0111 P11,
Escherichia
virus VT2phi 272, Escherichia virus TL-2011c, Escherichia virus P13374,
Escherichia virus Sp5.
[310] In one embodiment, the bacterial virus particles typically target E.
coil and include the
capsid of a bacteriophage selected in the group consisting of BW73, B278, D6,
D108, E, El, E24,
E41, F1-2, F1-4, F1-5, HI8A, Ff18B, i, MM, Mu, 025, Ph1-5, Pk, PSP3, PI, PID,
P2, P4, SI, W(p,
(pK13, cpl, p2, (p7, (p92, 7 A, 8(p, 9(p, 18, 28-1, 186, 299, HH-Escherichia
(2), AB48, CM, 04, 016,
DD-VI, E4, E7, E28, FII, F13, H, HI, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5,
ND6, ND-7, Ox-
I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, Ph1-1, RB42, RB43, RB49, RB69, S, Sal-I, Sal-
2, Sal-3, Sal-4,
Sal-5, Sal-6, 1023, 1045, Tull*-6, TuIP-24, Tull*46, TuIP-60, 12, 14, 16, 135,
al, 1, IA, 3, 3A,
31+, 5(p, 92660, 0F0103, HK620, J, K, KIF, m59, no. A, no. E, no. 3, no. 9,
N4, sd, 13, 17,
WPK, W31, AH, (p03888, (pK3, (pK7, (pK12, (pV-1, 004-CF, 005, 006, 007, pl,
p1.2, (p20, (p95,
(p263, (p1092, pl, pH, 08, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39,
42, 933W, NN-Escherichia
(1), Esc-7-11, A030, CVX-5, Cl, DDUP, ECI, E02, E21, E29, F1, F265, F275, Hi,
HK022, HK97,
HK139, HK253, HK256, K7, ND-I, PA-2, q, S2, T1, ), 130, 15, UC-I, w, 134, y2,
A, l)D326, (py,
006, 1)7, 010, (p80, x, 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, A06, A07,
A028, A043, A050,
A057, A081, A095, HK243, Kb, ZG/3A, 5, 5A, 21EL, H19-J and 933H.
Pharmaceutical or veterinary composition
[311] The present disclosure also provides a pharmaceutical or veterinary
composition
comprising the bacterial delivery vehicle as defined in the section "Bacterial
delivery vehicle"
above and a pharmaceutically acceptable carrier.
[312] Generally, for pharmaceutical use, the bacterial delivery vehicles may
be formulated as
a pharmaceutical preparation or composition comprising at least one bacterial
delivery vehicle
and at least one pharmaceutically acceptable carrier, diluent or excipient,
and optionally one or
more further pharmaceutically active compounds. Such a formulation may be in a
form suitable
for oral administration, for parenteral administration (such as by
intravenous, intramuscular or
subcutaneous injection or intravenous infusion), for topical administration,
for administration by
inhalation, by a skin patch, by an implant, by a suppository, etc. In a
particular embodiment, said
composition is for oral administration. Such administration forms may be
solid, semi-solid or
liquid, depending on the manner and route of administration. For example,
formulations for oral
administration may be provided with an enteric coating that will allow the
synthetic bacterial
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delivery vehicles in the formulation to resist the gastric environment and
pass into the intestines.
More generally, synthetic bacterial delivery vehicle formulations for oral
administration may be
suitably formulated for delivery into any desired part of the gastrointestinal
tract. In addition,
suitable suppositories may be used for delivery into the gastrointestinal
tract. Various
pharmaceutically acceptable carriers, diluents and excipients useful in
bacterial delivery vehicle
compositions are known to the skilled person
[313] The pharmaceutical or veterinary composition according to the disclosure
may further
comprise a pharmaceutically acceptable vehicle. A solid pharmaceutically
acceptable vehicle
may include one or more substances which may also act as flavouring agents,
lubricants,
solubilisers, suspending agents, dyes, fillers, glidants, compression aids,
inert binders,
sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
Suitable solid
vehicles include, for example calcium phosphate, magnesium stearate, talc,
sugars, lactose,
dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes
and ion exchange
resins.
[314] The pharmaceutical or veterinary composition may be prepared as a
sterile solid
composition that may be suspended at the time of administration using sterile
water, saline, or
other appropriate sterile injectable medium. The pharmaceutical or veterinary
compositions
disclosed herein may be administered orally in the form of a sterile solution
or suspension
containing other solutes or suspending agents (for example, enough saline or
glucose to make
the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate,
polysorbate 80 (oleate
esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and
the like. The
particles according to the disclosure can also be administered orally either
in liquid or solid
composition form. Compositions suitable for oral administration include solid
forms, such as pills,
capsules, granules, tablets, and powders, and liquid forms, such as solutions,
syrups, elixirs,
and suspensions. Forms useful for enteral administration include sterile
solutions, emulsions,
and suspensions.
[315] The bacterial delivery vehicles disclosed herein may be dissolved or
suspended in a
pharmaceutically acceptable liquid vehicle such as water, an organic solvent,
a mixture of both
or pharmaceutically acceptable oils or fats. The liquid vehicle can contain
other suitable
pharmaceutical additives such as solubilisers, emulsifiers, buffers,
preservatives, sweeteners,
flavouring agents, suspending agents, thickening agents, colours, viscosity
regulators,
stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral
and enteral
administration include water (partially containing additives as above, e.g.
cellulose derivatives,
preferably sodium carboxymethyl cellulose solution), alcohols (including
monohydric alcohols
and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.
fractionated coconut oil
and arachis oil). For parenteral administration, the vehicle can also be an
oily ester such as ethyl
oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile
liquid form compositions
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for enteral administration. The liquid vehicle for pressurized compositions
can be a halogenated
hydrocarbon or other pharmaceutically acceptable propellant.
[316] For transdermal administration, the pharmaceutical or veterinary
composition can be
formulated into ointment, cream or gel form and appropriate penetrants or
detergents could be
used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide
and
dimethylformamide.
[317] For transmucosal administration, nasal sprays, rectal or vaginal
suppositories can be
used. The active compounds can be incorporated into any of the known
suppository bases by
methods known in the art. Examples of such bases include cocoa butter,
polyethylene glycols
(carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with
other compatible
materials to modify the melting point or dissolution rate.
[318] In another particular embodiment, the present disclosure provides a
pharmaceutical or
veterinary composition as defined above for use to improve the effectiveness
of drugs. Indeed,
some bacteria of the microbiome, without being pathogenic by themselves, are
known to be able
to metabolize drugs and to modify them in ineffective or harmful molecules.
[319] In another particular embodiment, the disclosure provides a composition
that may further
comprise at least one additional active ingredient, for instance a prebiotic
and/or a probiotic
and/or an antibiotic, and/or another antibacterial or antibiofilm agent,
and/or any agent
enhancing the targeting of the bacterial delivery vehicle to a bacteria and/or
the delivery of the
payload into a bacteria.
[320] As used herein, a "prebiotic" refers to an ingredient that allows
specific changes, both in
the composition and/or activity in the gastrointestinal microbiota that may
confer benefits upon
the host. A prebiotic can be a comestible food or beverage or ingredient
thereof. A prebiotic may
be a selectively fermented ingredient. Prebiotics may include complex
carbohydrates, amino
acids, peptides, minerals, or other essential nutritional components for the
survival of the
bacterial composition. Prebiotics include, but are not limited to, amino
acids, biotin, fructo-
oligosaccharide, galacto-oligosaccharides, hemicelluloses (e.g., arabinoxylan,
xylan,
xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan
oligosaccharides, oligofructose-
enriched inulin, gums (e.g., guar gum, gum arabic and carrageenan),
oligofructose,
oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch),
trans-
galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple
pectin, and
rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea
fiber, corn bran, and
oat fiber) and xylooligosaccharides.
[321] As used herein, a "probiotic" refers to a dietary supplement based on
living microbes
which, when taken in adequate quantities, has a beneficial effect on the host
organism by
strengthening the intestinal ecosystem. Probiotic can comprise a non-
pathogenic bacterial or
fungal population, e.g., an immunomodulatory bacterial population, such as an
anti-inflammatory
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bacterial population, with or without one or more prebiotics. They contain a
sufficiently high
number of living and active probiotic microorganisms that can exert a
balancing action on gut
flora by direct colonisation. It must be noted that, for the purposes of the
present description, the
term "probiotic" is taken to mean any biologically active form of probiotic,
preferably including
but not limited to lactobacilli, bifidobacteria, streptococci, enterococci,
propionibacteria or
saccharomycetes but even other microorganisms making up the normal gut flora,
or also
fragments of the bacterial wall or of the DNA of these microorganisms. These
compositions are
advantageous in being suitable for safe administration to humans and other
mammalian subjects
and are efficacious for the treatment, prevention, of a disease or disorder
caused by bacteria
such as bacterial infection. Probiotics include, but are not limited to
lactobacilli, bifidobacteria,
streptococci, enterococci, propionibacteria, saccharomycetes, lactobacilli,
bifidobacteria, or
proteobacteria.
[322] The antibiotic can be selected from the group consisting of penicillins
such as penicillin
G, penicillin K, penicillin N, penicillin 0, penicillin V, methicillin,
benzylpenicillin, nafcillin, oxacillin,
cloxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin,
hetacillin, bacampicillin,
metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin,
temocillin, mezlocillin, and
piperacillin; cephalosporins such as cefacetrile, cefadroxil, cephalexin,
cefaloglycin, cefalonium,
cephaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone,
cefazolin, cefradine,
cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam,
cefmetazole,
cefotetan, cefoxitin, loracarbef, cefbuperazone, cefminox, cefotetan,
cefoxitin, cefotiam,
cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime,
cefmenoxime, cefodizime,
cefotaxime, cefovecin, cefpimizole, cefpodoxime, cefteram, ceftamere,
ceftibuten, ceftiofur,
ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, latamoxef,
cefclidine, cefepime,
cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef,
ceftobiprole, ceftaroline,
ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone,
cefetrizole, cefivitril,
cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide,
cefuracetime, and
nitrocefin; polymyxins such as polysporin, neosporin, polymyxin B, and
polymyxin E, rifampicins
such as rifampicin, rifapentine, and rifaximin; Fidaxomicin; quinolones such
as cinoxacin,
nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin,
ciprofloxacin, enoxacin,
fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin,
rufloxacin, balofloxacin,
grepafloxacin, levofloxacin, pazufloxacin, temafloxacin, tosufloxacin,
clinafloxacin, gatifloxacin,
gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin,
delafloxacin, nemonoxacin,
and zabofloxacin; sulfonamides such as sulfafurazole, sulfacetamide,
sulfadiazine,
sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine, sulfamethoxazole,
sulfamoxole,
sulfanitran, sulfadimethoxine, sulfametho-xypyridazine, sulfametoxydiazine,
sulfadoxine,
sulfametopyrazine, and terephtyl; macrolides such as azithromycin,
clarithromycin,
erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin,
kitasamycin, midecamycin,
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oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, and
roxithromycin; ketolides
such as telithromycin, and cethromycin; fluoroketolides such as solithromycin;
lincosamides
such as lincomycin, clindamycin, and pirlimycin; tetracyclines such as
demeclocycline,
doxycycline, minocycline, oxytetracycline, and tetracycline; aminoglycosides
such as amikacin,
dibekacin, gentamicin, kanamycin, neomycin, netilmicin, sisomicin, tobramycin,
paromomycin,
and streptomycin; ansamycins such as geldanamycin, herbimycin, and rifaximin;
carbacephems
such as loracarbef; carbapenems such as ertapenem, doripenem, imipenem (or
cilastatin), and
meropenem; glycopeptides such as teicoplanin, vancomycin, telavancin,
dalbavancin, and
oritavancin; lincosamides such as clindamycin and lincomycin; lipopeptides
such as daptomycin;
monobactams such as aztreonam; nitrofurans such as furazolidone, and
nitrofurantoin;
oxazolidinones such as linezolid, posizolid, radezolid, and torezolid;
teixobactin, clofazimine,
dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid,
pyrazinamide, rifabutin,
arsphenamine,chloramphenicol, fosfomycin, fusidic acid, metronidazole,
mupirocin,
platensimycin, quinupristin (or dalfopristin), thiamphenicol, tigecycline,
tinidazole, trimethoprim,
alatrofloxacin, fidaxomicin, nalidixic acid, rifampin, derivatives and
combination thereof.
Applications
[323] The present disclosure provides a method for in vivo delivery of a DNA
payload of interest
into a subject comprising, administering to said subject a pharmaceutical or
veterinary
composition as disclosed herein.
[324] Also provided are methods for treating a disease or disorder caused by
bacteria such as
bacterial infection using the bacterial delivery vehicles or compositions
disclosed herein. The
methods include administering a therapeutically efficient amount of bacterial
delivery vehicles or
compositions disclosed herein to a subject having a bacterial infection in
need of treatment.
[325] The present disclosure also provides the pharmaceutical or veterinary
compositions
disclosed herein or the bacterial delivery vehicles disclosed herein for use
in a method for
treating a disease or disorder caused by bacteria.
[326] Another object of the disclosure concerns providing the use of a
bacterial delivery vehicle
as described herein for the manufacture of a medicament intended for the
treatment of a disease
or disorder caused by bacteria.
[327] In some embodiments, the subject is a mammal. In some embodiments, the
subject is a
human.
[328] Said disease or disorder may be a bacterial infection, a metabolic
disorder or a pathology
involving bacteria of the human microbiome.
[329] The diseases or disorders caused by bacteria may be selected from the
group consisting
of abdominal cramps, acne vulgaris, acute epiglottitis, arthritis,
bacteraemia, bloody diarrhea,
botulism, Brucellosis, brain abscess, chancroid venereal disease, Chlamydia,
Crohn's disease,
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conjunctivitis, cholecystitis, colorectal cancer, polyposis, dysbiosis, Lyme
disease, diarrhea,
diphtheria, duodenal ulcers, endocarditis, erysipelothricosis, enteric fever,
fever,
glomerulonephritis, gastroenteritis, gastric ulcers, Guillain-Barre syndrome
tetanus, gonorrhoea,
gingivitis, inflammatory bowel diseases, irritable bowel syndrome,
leptospirosis, leprosy,
listeriosis, tuberculosis, Lady Windermere syndrome, Legionaire's disease,
meningitis,
mucopurulent conjunctivitis, multi-drug resistant bacterial infections, multi-
drug resistant
bacterial carriage, myonecrosis-gas gangrene, mycobacterium avium complex,
neonatal
necrotizing enterocolitis, nocardiosis, nosocomial infection, otitis,
periodontitis, phalyngitis,
pneumonia, peritonitis, purpuric fever, Rocky Mountain spotted fever,
shigellosis, syphilis,
sinusitis, sigmoiditis, septicaemia, subcutaneous abscesses, tularaemia,
tracheobronchitis,
tonsillitis, typhoid fever, ulcerative colitis, urinary infection, whooping
cough.
[330] The disease or disorder caused by bacteria may be a bacterial infection
selected from
the group consisting of skin infections such as acne, intestinal infections
such as esophagitis,
gastritis, enteritis, colitis, sigmoiditis, rectitis, and peritonitis, urinary
tract infections, vaginal
infections, female upper genital tract infections such as salpingitis,
endometritis, oophoritis,
myometritis, parametritis and infection in the pelvic peritoneum, respiratory
tract infections such
as pneumonia, intra-amniotic infections, odontogenic infections, endodontic
infections, fibrosis,
meningitis, bloodstream infections, nosocomial infection such as catheter-
related infections,
hospital acquired pneumonia, postpartum infection, hospital acquired
gastroenteritis, hospital
acquired urinary tract infections, and a combination thereof. In an
embodiment, the infection
according to the disclosure is caused by a bacterium presenting an antibiotic
resistance. In a
particular embodiment, the infection is caused by a bacterium as listed above
in the targeted
bacteria.
[331] The disease or disorder caused by bacteria may also be a metabolic
disorder, for
example, obesity and/or diabetes. The disclosure thus also concerns a
pharmaceutical or
veterinary composition as disclosed herein for use in the treatment of a
metabolic disorder
including, for example, obesity and/or diabetes. It further concerns a method
for treating a
metabolic disorder comprising administering a therapeutically efficient amount
of the
pharmaceutical or veterinary composition as disclosed herein, and the use of a
pharmaceutical
or veterinary composition as disclosed herein for the manufacture of a
medicament for treating
a metabolic disorder.
[332] The disease or disorder caused by bacteria may also be a pathology
involving bacteria
of the human microbiome. Thus, in a particular embodiment, the disclosure
concerns a
pharmaceutical or veterinary composition as disclosed herein for use in the
treatment of
pathologies involving bacteria of the human microbiome, such as inflammatory
and auto-immune
diseases, cancers, infections or brain disorders. It further concerns a method
for treating a
pathology involving bacteria of the human microbiome comprising administering
a
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therapeutically efficient amount of the pharmaceutical or veterinary
composition as disclosed
herein, and the use of a pharmaceutical or veterinary composition as disclosed
herein for the
manufacture of a medicament for treating a pathology involving bacteria of the
human
microbiome. Indeed, some bacteria of the microbiome, without triggering any
infection, can
secrete molecules that will induce and/or enhance inflammatory or auto-immune
diseases or
cancer development. More specifically, the present disclosure relates also to
modulating
microbiome composition to improve the efficacy of immunotherapies based, for
example, on
CAR-T (Chimeric Antigen Receptor T) cells, TIL (Tumor Infiltrating
Lymphocytes) and Tregs
(Regulatory T cells) also known as suppressor T cells. Modulation of the
microbiome
composition to improve the efficacy of immunotherapies may also include the
use of immune
checkpoint inhibitors well known in the art such as, without limitation, PD-1
(programmed cell
death protein 1) inhibitor, PD-L1 (programmed death ligand 1) inhibitor and
CTLA-4 (cytotoxic T
lymphocyte associated protein 4).
[333] In certain embodiments, the disease to be treated is cancer or a
proliferative disorder,
including but not limited to, breast cancer (e.g., triple negative breast
cancer, ER+ breast cancer,
or ER- breast cancer), basal cell carcinoma, skin cancer, lung cancer, small
cell lung cancer,
non-small cell lung cancer, brain cancer, medulloblastoma, glioma (including
glioblastoma,
oligodendroglioma, astrocytoma, ependymoma), neuroblastoma, colorectal cancer,
ovarian
cancer, liver cancer, pancreatic cancer (e.g., carcinoma, angiosarcoma,
adenosarcoma), gastric
cancer, gastroesophageal junction cancer, prostate cancer, cervical cancer,
bladder cancer,
head and neck cancer, lymphoma (e.g., mantle cell lymphoma, diffuse large B-
cell lymphoma),
removable solid tumors or solid tumors that cannot be removed by surgery,
locally advanced
solid tumors, metastatic solid tumors, leukemia (e.g., acute myeloid leukemia
(AML), acute
lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML)), or recurrent
or refractory
tumors.
[334] In one embodiment, the diseases to be treated include, but are not
limited to,
inflammatory or allergic diseases, including systemic anaphylaxis and
hypersensitivity disorders,
atopic dermatitis, urticaria, drug allergies, insect sting allergies, food
allergies (including celiac
disease and the like), and mastocytosis; inflammatory bowel diseases,
including Crohn's
disease, ulcerative colitis, ileitis, and enteritis; vasculitis, and Behcet's
syndrome; psoriasis and
inflammatory dermatoses, including dermatitis, eczema, atopic dermatitis,
allergic contact
dermatitis, urticaria, viral cutaneous pathologies including those derived
from human
papillomavirus, HIV or RLV infection, bacterial, flugal, and other parasital
cutaneous pathologies,
and cutaneous lupus erythematosus; asthma and respiratory allergic diseases,
including allergic
asthma, exercise induced asthma, allergic rhinitis, otitis media, allergic
conjunctivitis,
hypersensitivity lung diseases, and chronic obstructive pulmonary disease;
autoimmune
diseases, including arthritis (including rheumatoid and psoriatic), systemic
lupus erythematosus,
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type I diabetes, myasthenia gravis, multiple sclerosis, Graves' disease, and
glomerulonephritis;
graft rejection (including allograft rejection and graft-v-host disease),
e.g., skin graft rejection,
solid organ transplant rejection, bone marrow transplant rejection; fever;
cardiovascular
disorders, including acute heart failure, hypotension, hypertension, angina
pectoris, myocardial
infarction, cardiomyopathy, congestive heart failure, atherosclerosis,
coronary artery disease,
restenosis, and vascular stenosis; cerebrovascular disorders, including
traumatic brain injury,
stroke, ischemic reperfusion injury and aneurysm; fibrosis, connective tissue
disease, and
sarcoidosis, genital and reproductive conditions, including erectile
dysfunction; gastrointestinal
disorders, including gastritis, ulcers, nausea, pancreatitis, and vomiting;
neurologic disorders,
including Alzheimer's disease; sleep disorders, including insomnia,
narcolepsy, sleep apnea
syndrome, and Pickwick Syndrome; pain; renal disorders; ocular disorders,
including glaucoma;
and non-bacterial infectious diseases, including HIV.
[335] In some aspects, the disease to be treated may be an autoimmune disease
such as
autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune
neutropenia, autoimmunocytopenia, antiphospholipid syndrome, dermatitis,
gluten-sensitive
enteropathy, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart
disease, glomerulonephritis, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendo-
crinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune
Pulmonary
Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease,
rheumatic heart
disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,
autoimmune
inflammatory eye, autoimmune thyroiditis, hypothyroidism, systemic lupus
erythematosus,
discoid lupus, Goodpasture's syndrome, Pemphigus, Graves' Disease, Myasthenia
Gravis, and
insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic
purpura,
rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed
connective tissue
disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's
disease,
infertility, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome,
diabetes mellitus,
adrenergic drug resistance with asthma or cystic fibrosis, chronic active
hepatitis, primary biliary
cirrhosis, endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy
syndrome, urticaria,
atopic dermatitis, asthma, inflammatory myopathies, an inflammatory disorder,
a granulomatous
disorder, an atrophic disorder, or an alloimmune disease.
[336] The subject to be treated may have been diagnosed with, or may be at
risk of developing
an infection, a disorder and/or a disease preferably due to a bacterium.
Diagnostic methods of
such infection, disorder and/or disease are well known by the man skilled in
the art.
[337] In a particular embodiment, the infection, disorder and/or disease
presents a resistance
to treatment, preferably the infection, disorder or disease presents an
antibiotic resistance.
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[338] In a particular embodiment, the subject has never received any treatment
prior to the
administration of the delivery vehicles according to the invention or of the
pharmaceutical or
veterinary composition according to the invention.
[339] In a particular embodiment, the subject has already received at least
one line of
treatment, preferably several lines of treatment, prior to the administration
of the delivery
vehicles according to the invention or of the pharmaceutical or veterinary
composition according
to the invention.
[340] Preferably, the treatment is administered regularly, preferably between
every day and
every month, more preferably between every day and every two weeks, more
preferably
between every day and every week, even more preferably the treatment is
administered every
day. In a particular embodiment, the treatment is administered several times a
day, preferably 2
or 3 times a day, even more preferably 3 times a day.
[341] The duration of treatment with delivery vehicles according to the
invention or with the
pharmaceutical or veterinary composition according to the invention, is
preferably comprised
between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still
more preferably
between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. In
a particular
embodiment, the duration of the treatment is of or about 1 week.
Alternatively, the treatment may
last as long as the infection, disorder and/or disease persists.
[342] The form of the pharmaceutical or veterinary compositions, the route of
administration
and the dose of administration of delivery vehicles according to the invention
or of
pharmaceutical or veterinary composition according to the invention can be
adjusted by the man
skilled in the art according to the type and severity of the infection (e.g.
depending on the bacteria
species involved in the disease, disorder and/or infection and its
localization in the patient's or
subject's body), and to the patient or subject, in particular its age, weight,
sex, and general
physical condition.
[343] Particularly, the amount of delivery vehicles according to the invention
or of
pharmaceutical or veterinary composition according to the invention, to be
administered has to
be determined by standard procedure well known by those of ordinary skills in
the art.
Physiological data of the patient or subject (e.g. age, size, and weight) and
the routes of
administration have to be taken into account to determine the appropriate
dosage, so as a
therapeutically effective amount will be administered to the patient or
subject.
[344] For example, the total amount of delivery vehicles according to the
invention for each
administration is between 104 and 1015 delivery vehicles.
[345] In a particular embodiment, in the treatment methods or uses, said
composition or
bacterial delivery vehicle is administered orally.
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[346] Some bacteria of the microbiome can also secrete molecules that will
affect the brain,
such as serotonin and melatonin for use in the treatment of depression,
dementia or sleep
disorder.
[347] Therefore, a further object of the disclosure is a method for
controlling the microbiome of
a subject, comprising administering an effective amount of the pharmaceutical
or veterinary
composition as disclosed herein in said subject.
[348] In a particular embodiment, the disclosure also relates to a method for
personalized
treatment for an individual in need of treatment for a disease or disorder
such as bacterial
infection comprising: i) obtaining a biological sample from the individual and
determining a group
of bacterial DNA sequences from the sample; ii) based on the determining of
the sequences,
identifying one or more pathogenic bacterial strains or species that were in
the sample; and iii)
administering to the individual a pharmaceutical or veterinary composition
according to the
disclosure capable of recognizing each pathogenic bacterial strain or species
identified in the
sample and to deliver the packaged payload.
[349] In an embodiment, the biological sample comprises pathological and non-
pathological
bacterial species, and subsequent to administering the pharmaceutical or
veterinary composition
according to the disclosure to the individual, the amount of pathogenic
bacteria on or in the
individual are reduced, but the amount of non-pathogenic bacteria is not
reduced.
[350] In another particular embodiment, the disclosure concerns a
pharmaceutical or
veterinary composition according to the disclosure for use to improve the
effectiveness of drugs.
Indeed, some bacteria of the microbiome, without being pathogenic by
themselves, are known
to be able to metabolize drugs and to modify them in ineffective or harmful
molecules.
[351] In another aspect, the methods and compositions described herein provide
long term
stable expression of a gene of interest in the microbiome of a host. In such
an instance, the
delivery vehicle comprises a nucleic acid molecule encoding the gene of
interest wherein the
nucleic acid is engineered to either integrate into the bacterial chromosome
or, alternatively,
stably replicate within the targeted microbiome of the host. Once delivered
into the bacteria of
interest, i.e., the microbiome, the gene of interest will typically be
expressed. In a particular
embodiment, the disclosure concerns the in-situ bacterial production of any
compound of
interest, including therapeutic compound such as prophylactic and therapeutic
vaccine for
mammals. The compound of interest can be produced inside the targeted
bacteria, secreted
from the targeted bacteria or expressed on the surface of the targeted
bacteria. In a more
particular embodiment, an antigen is expressed on the surface of the targeted
bacteria for
prophylactic and/or therapeutic vaccination.
[352] The present disclosure also provides a method for reducing the amount of
virulent and/or
antibiotic resistant bacteria in a bacterial population comprising contacting
the bacterial
population with an efficient amount of the bacterial delivery vehicle as
defined in the section
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"Bacterial delivery vehicle" above. The present disclosure further provides
the bacterial delivery
vehicles as defined in the section "Bacterial delivery vehicle" above, for use
in a method for
reducing the amount of virulent and/or antibiotic resistant bacteria in a
bacterial population, in
particular in the treatment of a bacterial infection typically due to virulent
and/or antibiotic
resistant bacteria. Another object of the disclosure provides the use of the
bacterial delivery
vehicle as defined in the section "Bacterial delivery vehicle" above for the
manufacture of a
medicament intended for reducing the amount of virulent and/or antibiotic
resistant bacteria in a
bacterial population, in particular for the treatment of bacterial infection
typically due to virulent
and/or antibiotic resistant bacteria.
[353] The present disclosure also relates to a non-therapeutic use of the
bacterial delivery
particles. For instance, the non-therapeutic use can be a cosmetic use or a
use for improving
the well-being of a subject, in particular a subject who does not suffer from
a disease.
Accordingly, the present disclosure also relates to a cosmetic composition or
a non-therapeutic
composition comprising the bacterial delivery particles of the disclosure.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: Description
1 Insertion site sequence SAG DAS
2 Insertion site sequence ADAKKS
3 Insertion site sequence MDETNR
4 Insertion site sequence SASAAA
Insertion site sequence GAGENS
6 GSATDVMIQL sequence
7 GSATDVMIQLA sequence
8 Lambda STF amino acid sequence
9 STF-V10-[FA] amino acid sequence
STF-V10-[AAI-1] amino acid sequence
11 STF-V10-[Helix] amino acid sequence
12 K5 amino acid sequence
13 K5 5.0 amino acid sequence
14 K5 5.1 amino acid sequence
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15 STF-V10 amino acid sequence
16 V10 amino acid sequence
17 STF-V10-[FA] DNA sequence
18 STF-V10-[AAH] DNA sequence
19 STF-V10-[Helix] DNA sequence
20 K5 5.0 DNA sequence
21 K55.1 DNA sequence
22 Lambda gpJ amino acid sequence
23 H591 amino acid sequence
24 H591 DNA sequence
25 Z2145 amino acid sequence
26 Z2145 DNA sequence
27 1A2 amino acid sequence
28 1A2 DNA sequence
29 A8 amino acid sequence
30 A8 DNA sequence
31 gpH 1AI amino acid sequence
32 Lambda-K5 amino acid sequence
33 Payload p1392 plasmid sequence
34 helical bundle 1 and linker from STF protein from Escherichia
phage ZG49
amino acid sequence
35 recoded helical bundle 1 and linker from STF protein from
Escherichia
phage ZG49 DNA sequence
36 helical bundle 2 and linker from STF protein from Escherichia
phage ZG49
amino acid sequence
37 recoded helical bundle 2 and linker from STF protein from
Escherichia
phage ZG49 DNA sequence
38 K5 9.0 amino acid sequence
39 K5 9.0 DNA sequence
40 K5 9.1 amino acid sequence
41 K59.1 DNA sequence
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42 payload p1900 plasmid sequence
43 candidate STF protein from Escherichia phage ZG49 amino acid
sequence
44 candidate STF protein from Escherichia phage ZG49 DNA sequence
45 payload p775 plasmid sequence
46 primase on from the PIC1 of the Escherichia coli strain CFT073
47 restriction site
48 Primase on deltaGAAABCC
49 Primase on devoid of restriction sites
50 PIC1 primase-helicase amino acid sequence
51 PIC! primase-helicase DNA sequence
EXAMPLES
EXAMPLE 1
[354] It has been shown that a chimera between lambda STF and V10 STF
(originating from
a prophage found in 0157 strains), said chimera being of sequence SEQ ID NO:
15, is able to
target 0157 strains with high efficiency in vitro by recognizing and degrading
the 0157 antigen
group IV capsule. However, initial in vivo experiments showed that lambda
packaged phagemids
containing the V10 chimeric STF did not deliver with high efficiency into 0157
strains colonizing
the mouse gut. Efficiencies of delivery in this mouse model were, on average,
20% and the
delivery was not improved by increasing the dosage given to the mouse (M01).
[355] One possible reason for this observation was that the chimeric lambda
particles
containing V10 fusions were stable in in vitro conditions, where delivery and
killing experiments
were done in the presence of known reagents (for instance, LB), but lost part
of their activity
once they passed through the mouse gut.
[356] It had been observed that wild-type lambda particles are able to pass
and replicate in the
gut suggesting that some part of the engineering process to generate the
lambda-V10 fusion
had rendered it at least less stable and partly susceptible to degradation in
in vivo conditions.
Apart from the lambda STF-V10 fusion, the lambda particles used in these
experiments have
also been engineered at the gpJ level to modify its primary receptor, and
contain the 1A2 gpJ
variant (and are thus called herein 1A2-V10 particles). Thus, it was possible
that either the 1A2
gpJ variant and/or the STF-V10 fusion were the sources of reduced stability in
in vivo conditions.
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[357] In vitro assays were set up to differentiate between 1A2 gpJ activity
and STF-V10 activity
based on the fact that, for some strains, the presence of a functional STF is
dispensable for
injection, as is the case for the MG1655 K-12 strain. Since the 1A2 gpJ
variant recognizes the
OmpC receptor of 0157 strains, but not that of MG1655, an MG1655 variant was
engineered in
which the OmpC receptor was replaced to encode that of the 0157 variant. This
strain was
called MG1656-0mpC0157. On the other hand, efficient delivery in 0157 strains
is completely
dependent on the presence of a functional STF containing V10. Hence, by
exposing the 1A2-
V10 packaged phagemids to different conditions and evaluating the gpJ versus
STF-V10 activity
in vitro, it was possible to determine which part of the packaged phagemid was
unstable.
[358] The 1A2-V10 packaged phagemids were then exposed to simulated intestinal
fluid (SIF)
in the presence or absence of pancreatin (which contains the digestive enzymes
trypsin and
chymotrypsin) and bile salts. Specifically, packaged phagemids were produced,
diluted 1:100 in
the buffer of choice and incubated at 372C for 3 hours. After that, the
packaged phagemids were
directly titrated on MG1656-OmpC0157 and H10 (0157)-delta-stx strains. As a
control, the wild-
type lambda packaged phagemid produced with CYC3 strains was also exposed to
the same
conditions. H10-delta-stx is a variant of 0157 strain for which the stx gene
has been deleted.
Briefly, the wild-type H10 strain was transduced with packaged lambda
phagemids containing a
lambda-V10 STF chimera and a packaged circuit encoding a Cpf1 nuclease
programmed to
target the stx2 gene. After transduction, survivor colonies were checked by
PCR to verify the
presence or absence of the stx gene and only colonies with stx gene deleted
were kept.
[359] As can be seen in Figure 1, the wild-type lambda particle produced with
CYC3 strains
was stable under any conditions, as the titers remained the same across all
experiments.
However, for the 1A2-V10 variant, a constant gpJ activity (central bars in
Figure 1) was
observed, which indicates that this gpJ variant was not degraded in the
presence of pancreatin.
Finally, the titers of the 1A2-V10 dropped by a factor of 2 log when titrated
in H10-delta-stx
(0157) strains only in the presence of pancreatin. Bile salts by themselves
did not affect the
activity of the packaged phagemids. These results clearly demonstrate that the
STF-V10
chimera is at least partially degraded in the presence of pancreatin.
[360] It was hypothesized that the source of reduced stability was not in the
V10 moiety itself,
but in the way the fusion with the lambda STF was generated. Further, it was
hypothesized that
although no linker amino acids were inserted in the initial lambda STF-V10
chimera, the context
of the fusion was not natural, and hence, had not been selected for stability
in the presence of
proteolytic enzymes. To test this hypothesis, two types of lambda STF-V10
chimeras were
generated: the first type contains point mutations in phenylalanine (F) and
lysine (K) residues
present in the fusion point between lambda STF and V10 STF (Figure 2); for the
second type,
a more detailed structural analysis was performed. Structural homology
analyses with the
original V10 fusion showed a crystallized STF with high identity to the V10
moiety (PDB ID:
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5W6S): this STF contains a short helix at its N-terminus which has a homolog
in V10, but that
was not included in the original lambda STF-V10 chimera. The helix forms a
very tight bundle
that "fastens" the domain right after it in the crystal structure. Based on
the delivery efficiency
results that were obtained with the original lambda STF-V10 version, this
helix may not be
important for activity but it may be important for stability since it may
confer a proper folding
where exposed trypsin- and chymotrypsin-accessible residues are buried (Figure
2).
[361] Accordingly, three lambda-STF-V10 fusion variants were constructed: V10-
[FA] (SEQ ID
NO: 9), where a lysine (K) residue was exchanged by an alanine (A); V10-[AAH]
(SEQ ID NO:
10), where an FKF tripeptide was exchanged to AAH tripeptide; and V10-Helix
(SEQ ID NO: 11),
where the short 10-amino acid helix bundle GSATDVMIQLA (SEQ ID NO: 7) was
included as
part of the chimeric protein just after the insertion site. The insertion site
with the lambda STF,
GAGENS (SEQ ID NO: 5), was not changed for any of the variants.
[362] The three variants were then exposed to buffer at different pH values
(5.0 and 6.8) in the
presence or absence of pancreatin as detailed for the original lambda STF-V10
fusion above.
As can be seen in Figure 3, all variants showed some degree of resistance to
pancreatin
treatment: the V10-[FA] and V10-[AAH] variants showed between 1 and 1.5 log
higher particle
levels than the original V10 counterpart, although the stability was not
complete and was
dependent on pH. However, the V10-Helix variant showed an apparent complete
resistance to
digestive proteases at any pH tested. Taken together, the results showed that
one can engineer
lambda STF-V10 variants that are resistant to digestive proteases by only
engineering the linker
region, and that are good candidates for in vivo use, with V10-Helix showing
highly positive
results in vitro.
[363] In vivo studies were next conducted. It was difficult to deliver 0157
strains in vivo at a
decent efficiency (max 40%, but typically under 20%) and interestingly, the
delivery was not
improved by increasing the MOI administered to the mouse. However, delivery
was observed
with the same vector while using a strain deleted for the 0157 antigen (AwaaJ
mutants). Based
on this result, it was possible that the V10 activity somehow may not survive
the transit through
the GIT.
[364] In vivo assays were conducted to measure the kinetics of shedding of
packaged
phagemids with 1A2 gpJ and chimeric lambda STF-V10 after oral administration
to streptomycin-
treated, uncolonized BALB/c mice as well as the residual V10 activity. The
specific V10 activity
was evaluated by comparing transduction efficiencies on H10Astx, where 1A2 and
V10 are both
needed and MG-ompC 0157 where only 1A2 is required. Packaged phagemids were
produced
at high titer and given to 3 mice in sucrose-bicarbonate buffer to reduce the
stomach acidity and
to help packaged phagemids to reach the intestine. Stool samples were
collected at TO, T2h,
T4h, T6h and T8h, and resuspended in PBS. After centrifugation, supernatants
containing shed
packaged phagemids were used in transduction assays against H10Astx and MG-
ompC 0157.
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[365] Interestingly, as can be seen in Figure 4, the initial dose of packaged
phagemid
contained approximately 10% of the particles with V10 activity. Most of the
1A2 activity could be
recovered between 6 to 8 hours after oral gavage, which indicates that this
gpJ variant is not
degraded after transit through the entire Gastrointestinal Tract (GIT).
However, the estimated
titers measuring the V10 activity were very low. Less than 1% of the recovered
packaged
phagemids kept their V10 functionality. This result indicates that at least
90% of the packaged
phagemid with 1A2 gpJ and chimeric lambda STF-V10 lose their V10 activity in
the GIT. The
presence of numerous proteolytic enzymes (trypsin and chymotrypsin) secreted
by the pancreas
in the gut may be responsible for this degradation. This experiment finally
demonstrates that
1A2-V10 can survive through the GI tract but loses an important part of its
V10 activity which
would explain why one cannot deliver in 0157 strains at high efficiency.
[366] Following on from this experiment and the result of in vitro stability
testing of 3 new
lambda-STF-V10 fusion variants, 2 variants that seemed to better resist the
digestive proteases
were used: lambda-STF-V10-[FA] and lambda-STF-V10-[Helix], disclosed above.
Indeed, in
vitro experiments have shown that these packaged phagemids (also called
eligobiotics or EB)
seemed able to resist in pancreatin-containing medium at least lh without
losing their capacity
to deliver into strains where V10 activity is required. This was especially
true for the lambda-
STF-V10-[Helix] variant. Then, in the exact same conditions as with the
original 1A2-V10, the
inventors assessed the residual activity of V10 after passage through the
entire GI tract of
uncolonized BALB/c mice.
[367] As can be seen on Figure 5, V10 activity of the variant 1A2-V10-[FA] was
approximately
at 1% after passage through the gut. As opposed to this observation, the new
1A2-V10-[Helix]
showed a V10 activity approximately similar to the total activity of this
packaged phagemid after
passage through the GIT. These data indicate that 1A2-V10-[Helix] could
perform optimally in
vivo due to the high stability of its V10 activity (as opposed to the original
version of 1A2-V10).
To further confirm this stability, a simplified pharmacokinetics study was
conducted in mice,
where the shedding of the 1A2-V10-[Helix] over time was observed, following
oral gavage of
uncolonized BALB/c mice with a single dose of this packaged phagemid
(administered as a 1:1
mixture with a sucrose/bicarbonate buffer).
[368] As shown in Figure 6, the STF activity (required to enter into H10 but
not into MG1656)
was just as stable over time as the Tip/overall capside functionality, as
indicated by the identical
pattern of shedding in the stool.
[369] In another experiment, the in vivo delivery of the two new versions
through plasmid curing
was studied. The lambda-STF-V10-[Helix] and lambda-STF-V10-[FA] packaged
phagemids
were administered (2 doses, 6h apart) targeting one part of the pRFP plasmid
into mice
colonized with H10Astx/pRFP. Assuming that the payload is fully effective
(100% cutting efficacy
once expressed in the cell), the delivery can be calculated as the ratio of
bacteria that lost the
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target plasmid over the total number of bacteria. Practically, the plasmid
carries a kanamycin
resistance gene; this makes it easier to check for colonies that kept versus
lost the plasmid by
simply patching streptomycin-resistant bacteria onto Kan plates.
[370] As can be seen on Figure 7, the curing efficiency was great with this
mixture of packaged
phagemids as most of the mice displayed a curing percentage of 80% or more (9
mice out of
10). Even though a great peak was observed after a single administration at t
= 6h, the peak of
curing efficacy was higher at 24 hours post-treatment likely reflecting the
interest of a second
administration, although this may be due to transit time variations between
animals. Another
interesting observation is that pRFP curing (i.e., sensitivity to kanamycin)
was still visible at T24h
and T48h whereas payload delivery (i.e., resistance to chloramphenicol) had
strongly
decreased. This indicates that the curing method could give a more stable view
of
delivery/nuclease efficacy over time. The results clearly demonstrate that the
mixture of new
packaged phagemids tested is much more capable of targeting strains of
interest in the mouse
intestine.
[371] In order to optimize for phagemids, PCRs were conducted on several
clones from the
feces to discriminate between lambda-STF-V10-[Helix] and lambda-STF-V10-[FA]:
out of 38
tested clones, 71% had received the payload from lambda-STF-V10-[Helix],
indicating that this
version was significantly efficient under in vivo conditions.
[372] According to previous results, a decolonization experiment in vivo of
the STEC strain
H1OWT with the new mutant 1A2-V10-[Helix] was conducted. In order to avoid
colonization
rebound immediately after treatment with packaged phagemids, it was decided to
remove the
antibiotic pressure (streptomycin) that was used to clear and maintain a niche
for
Enterobacteriaceae in the gut of mice with conventional specific-pathogen-free
flora. Mice were
treated with 5 doses of the packaged phagemid, 2 days apart, and compared with
a control
group treated with 5 doses of buffer (sucrose Bicarbonate).
[373] As can be observed in Figures 8 and 9 on the control group, the
colonization was not
totally stable overtime. A slow decrease day after day can be seen from D6 to
D12. However,
the buffer did not seem to have an impact on the colonization level. In
contrast, the colonization
level of the STEC strains presented a great response to treatment. Indeed, a 2
logs reduction
was observed after the first dose and more than 3 logs after the second for 4
mice out of 5. After
the full 5-dose regimen (D7), a total of 4 logs of killing was obtained.
Interestingly, no rebound
of the colonization was observed after the last treatment.
[374] To check for a potential resistant population to the packaged phagemids
(natural or
acquired) at the end of the experiment, surviving colonies on D7/D8 were
patched and a
transduction experiment was carried out. Interestingly, no resistance (entry
or nuclease) was
observed in this experiment. Taken together, the results described herein show
an increased
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efficacy of variants, such as the variant 1A2-V10-[Helix] to decolonize STEC
strains from the
mouse gut.
EXAMPLE 2
[375] To test if the approach followed with the lambda-STF-V10 chimeric STF in
Example 1
above was generalizable to other STF chimeras, a second set of experiments was
performed.
In this case, a functional chimeric STF was engineered between lambda STF and
the K5
tailspike, called lambda-K5 (SEQ ID NO: 37) which has been described in the
literature to infect
K5-encapsulated E. coil strains and for which a crystal structure is available
[11]. The same
approach as for lambda-V10 chimera was followed, including the insertion point
in the lambda
STF protein (GAGENS (SEQ ID NO: 5)). In this case, the readout strain for K5
STF activity was
LMR 503 and the readout for gpJ activity was MG1656-OmpC0157, as explained
before.
Packaged phagemids harboring the 1A2 gpJ (SEQ ID: 27) and the lambda-K5 STF
were
produced and titrated in both LMR 503 or MG1656-OmpC0157 after treatment with
or without
pancreatin at pH 6.8.
[376] As can be seen in Figure 10, although the lambda-K5 STF chimera was
completely
functional as measured by its ability to inject into the LMR 503 strain in
PBS, it was not very
stable in the presence of pancreatin, showing up to 4-log loss in the number
of functional
particles. This was similar to what was observed for the lambda-STF-V10
chimeric STF.
[377] Next, the crystal structure was analyzed for the original K5 STF (PDB
ID: 2X3H) and it
was observed that it also contained a three helical bundle at its N-terminus.
However, as
opposed to the V10 structure, the helical bundle of K5 was capped by a turn,
which in the
lambda-K5 STF was directly at the fusion point. It was hypothesized that this
non-natural
insertion point may be the cause for the pancreatin reduced stability
observed. To test this
hypothesis, several lambda-K5 variants were constructed in which the fusion
point was modified
to contain different versions of the helical bundle.
= Lambda K5 5.0 (SEQ ID NO: 13): contains part of the helical bundle from
V10
(GSATDVMIQL (SEQ ID NO: 6)) fused to the K5 STF without its original helical
bundle
= Lambda K5 5.1 (SEQ ID NO: 14): contains the helical bundle from V10
(GSATDVMIQLA
(SED ID NO: 7)) fused to the K5 STF without its original bundle
[378] Packaged phagemids harboring the 1A2 gpJ and each of the K5 helix
chimeras were
produced and titrated on MG1656-OmpC0157 or LMR 503, as explained above.
[379] Figures 11 and 12 show that the variants containing V10 helix versions
K5 5.0 and K5
5.1 were mostly resistant to pancreatin treatment, as there was only 1 log
loss compared to other
STF fusions. It is also important to note that no functional differences in
terms of titers were
observed for any of the K5 variants constructed, which suggests a high degree
of flexibility in
terms of linkers to be used when creating non-homologous STF chimeras.
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[380] It has thus been shown that there was no correlation between function
(injection in a
given strain) and stability, and that the latter was dependent on the amino
acid content of the
fusion point. Additionally, the inventors showed that the sequence
GSATDVMIQL(A) (SEQ ID
NO: 6 and 7) originating from V10 Helix can be used as a pancreatin-resistant
linker even in
proteins that contain no homology to V10 STF (K5 STF) and protect the new
chimera from
degradation by pancreatin.
EXAMPLE 3
[381] Alternative pancreatin-resistant linkers conferring stability to a
lambda STF-K5 chimera
were designed from a STF protein having homology, at its C-terminal portion,
with the C-terminal
portion of the K5 STF starting at amino acid G62, namely candidate STF protein
from
Escherichia phage ZG49 (SEQ ID NO: 43 and SEQ ID NO: 44).
[382] An analysis of this ZG49 STF protein using HHPRED software (Soding et
al. (2005)
Nucleic Acids Res. 33:W244-8) showed that it contains a helical bundle from
amino acid 212 to
amino acid 217. This helical bundle was included in the linkers designed by
the inventors. More
particularly, these linkers comprise the amino acid sequence located between
amino acids G210
or D211 and amino acid E272 of the ZG49 phage STF protein. They are typically
of sequence
SEQ ID NO: 34 or SEQ ID NO: 36.
[383] Two chimeric STFs were then built that contain the N-terminus of Lambda
STF up to
amino acid sequence GAGENS (SEQ ID NO: 5), followed by the linker designed
above of
sequence SEQ ID NO: 34 or SEQ ID NO: 36, and followed by the K5 moiety
starting from position
G62. The DNA sequences of the designed linkers were recoded for expression in
Escherichia
coil and were respectively of sequence SEQ ID NO: 35 and SEQ ID NO: 37. The
two chimeric
STFs were called K5 9.0 (for linker starting at position G210, SEQ ID NO: 38
and SEQ ID NO:
39) and K5 9.1 (for linker starting at position D211, SEQ ID NO: 40 and SEQ ID
NO: 41) and
only differ in the presence or absence, respectively, of a glycine at the
start of the linker.
[384] The production and pancreatin tests of both chimeric STFs were done as
shown in
Examples 1 and 2, and showed that the use of a linker designed from a STF
protein having
homology at this C-terminal portion with the K5 STF also provided pancreatin
resistance to the
chimeric STFs, and even improved the pancreatin resistance of the chimera as
compared to K5
5.0 and K5 5.1 (Figure 15).
[385] Finally, in vivo assays were performed to attempt decolonization of the
LMR 503 strain,
which should be targeted in the gut only if the chimeric STF is resistant to
proteolytic enzymes,
as has been shown in Example 2. To do this, 10 BALB/c mice were treated with
streptomycin
and colonized with strain LMR 503. An Eligobiotic harboring the A8 gpJ and
the chimeric K5
9.1 STF was produced carrying a plasmid (p775, SEQ ID NO: 45) encoding a
nuclease and a
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guide targeting the ctx gene found in strain LMR 503. The decolonization assay
was identical
to that described for strain H1OWT, following a single dose of Eligobiotic
(Figure 16).
[386] A 2.6 log median reduction in strain levels was observed after treatment
with
Eligobiotic , which shows that the engineering of the K5 9.1 STF was
successful, and that K5
9.1 STF was able to withstand proteolytic degradation in the mouse gut.
[387] The inventors thus showed that other linkers could be designed to confer
pancreatin
resistance to chimeric RBP proteins. In particular, it is herein shown that
the sequences SEQ ID
NO: SEQ ID NO: 34 and SEQ ID NO:36 designed from the ZG49 phage STF protein
can be
used as a pancreatin-resistant linker to protect chimera comprising a lambda
STF N-terminal
portion and a K5 STF C-terminal portion from degradation by pancreatin.
EXAMPLE 4
[388] To evaluate the effect of DNA payload size on the number of payloads
packaged in
Eligobiotics , 3 different payloads were used to produce Eligobiotics as
summarized in Table
1.
Table 1: Batches of Eliqobiotics produced
Eligobiotic code / batch number Payload Size (kb)
eb512 / EB003-DS-008 p1085 12.125
eb393 / EB003-DS-009 p779 12.428
eb827 / EB003-DS-011 p1392 11.615
[389] After fermentation, lysis (3 h incubation at 37 C with 0.1% Triton X-
100, 2000 U/L
Benzonase) and clarification on a Zeta Plus Capsule (3M), the Eligobiotics
were purified by
anion exchange chromatography on a Sartobind Q capsule (Sartorius). This
initial purification
was followed by a buffer exchange and concentration step by tangential flow
filtration on a
Pellicon 2 minicassette Biomax 300kDa (Millipore). A final polishing step of
size exclusion
chromatography on Sepharose 6FF resin (GE Healthcare) was performed to yield
the purified
Eligobiotics .
[390] Analysis of the Eligobiotics 's DNA content was performed by analytical
ultracentrifugation in a Beckman Coulter Optima AUC using an AN50Ti rotor at 6
krpm. The
sedimentation coefficients of different particles present in solution for each
EB batch were
extracted from sedimentation velocity data (acquired at 260 and 280 nm).
[391] Based on the molecular weight calculated from their sedimentation
coefficient and their
260/280 nm ratios, the different populations of particles detected could be
separated as
Eligobiotics containing either 3 copies (centered on 290 S) or 4 copies
(centered on 330-340
S) of the payload (Figure 13).
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[392] Important differences were observed between Eligobiotics depending on
the size of the
packaged payload. Although Eligobiotics packaging the smaller p1392 (11.615
kb) yielded
almost exclusively particles containing 4 copies of the payload, small
increases (up to 800 bp)
in the size of the payload correlate with a shift towards packaging 3 copies.
As such,
Eligobiotics produced with p779 (12.428 kb) packaged preferentially 3 copies
of the payload
while approximately a third of the particles contained 4 copies (Figure 14).
[393] Thus, it appears that p1392 is close to an ideal size to package
exclusively 4 copies of
payload in Eligobiotics particles, yielding an homogenous population.
Increasing the size of the
payload compared to p1392 generates more heterogeneous Eligobiotics
populations, with
increasing proportions of particles containing 3 copies of payload. From this
dataset, it appears
that there is a lower limit for concatemer packaging close to 36 kb, as
described in the literature
[28]. p1085, with a size of 12.125 kb, could package 3 copies per head (36.375
kb) or 4 copies
per head (48.5 kb), although the 4 copies species is preferred as seen in
Figure 14. Increasing
the size to 12.428 kb would allow packaging of 3 copies per head (37.284 kb)
and 4 copies per
head (49.712 kb); in this case, 4 copies are preferred. From these two data
points, the inventors
inferred that the lower limit for packaging is indeed around 36 kb but with a
lower efficiency.
Increasing the size just by 909 bp completely shifts the packaged species to 4
copies: the limit
for optimal efficiency of packaging, probably driven by a pressure signal in
the capsid, lies within
these two sizes. Finally, the 11.615 kb payload packages virtually only 4
copies per head (46.46
kb), as the 3-copy species is slightly below the packaging limit, even at low
efficiency (34.845
kb).
[394] From these data, it can also be predicted which sizes would give
packaging of single and
multimeric species, as shown below in Tables 2 and 3. Smaller sizes yielding
single packaged
species are generally preferred for several reasons, including ease of
manipulation and lower
probability of introducing unwanted restriction sites. Finally, sizes that
allow for very efficient
packaged species that are not too small (26-39 kb) or too large (50-51 kb) are
also preferred in
some cases as it has been shown that the amount of DNA present in the capsid
may alter the
packaging and stability of the particles due to intracapsid pressure [29]-
[30]. Finally, sizes that
are large enough to allow for production of packaged phagemids at high titer
are also more
particularly preferred.
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Table 2: Predicted number of concatemers packaged in a capsid depending on the
monomer
size.
Number of copies in the concatemer
Plasm id 2 3 4 5 6 7 8 9 10
11
size (kb)
3 6 9 12 15 18 21 24 27 30 33
4 8 12 16 20 24 28 32 36 40 44
5 10 15 20 25 30 35 40 45 50 55
6 12 18 24 30 36 42 48 54 60 66
7 14 21 28 35 42 49 56 63 70 77
8 16 24 32 40 48 56 64 72 80 88
9 18 27 36 45 54 63 72 81 90 99
10 20 30 40 50 60 70 80 90 100 110
Single conformation possible, 4 copies 11 22 33 44 55
66 77 88 99 110 121
12 24 36 48 60 72 84 96 108 120 132
Single conformation possible 13 26 39 52 65 78 91
104 117 130 143
Single conformation possible 14 28 42 56 70 84 98
112 126 140 154
Single conformation possible 15 30 45 60 75 90
105 120 135 150 165
Single conformation possible 16 32 48 64 80 96
112 128 144 160 176
Single conformation possible, high limit 17 34 51 68 85 102
119 136 153 170 187
Single conformation possible 18 36 54 72 90
108 126 144 162 180 198
Single conformation possible 19 38 57 76 95 114 133 152 171
190 209
Single conformation possible 20 40 60 80 100 120 140 160 180 200 220
Single conformation possible 21 42 63 84 105 126 147 168 189 210 231
Single conformation possible 22 44 66 88 110 132 154 176 198 220 242
Single conformation possible 23 46 69 92 115 138 161
184 207 230 253
Single conformation possible 24 48 72 96 120 144 168 192 216 240 264
Cells with heavy dark borders and in bold represent better species, cells with
thin borders and
non-bolded represent species either too small or too large for optimal
packaging. The lower and
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higher limits for efficient packaging have been set to 36 kb and 51 kb,
respectively.
Table 3: Predicted number of concatemers packaged in a capsid depending on the
monomer
size between 9 and 13 kb.
Number of copies in the concatemer
Plasmid size (kb) 2 3 4 5 6
9 18 27 36 45 54
9.25
18.5 27.75 37 46.25 55.5
9.5 19 28.5 38 47.5
57
9.75
19.5 29.25 39 48.75 58.5
20 30 40 50 60
Single conformation possible, 4 copies 10.25 20.5 30.75
41 51.25 61.5
Single conformation possible, 4 copies 10.5 21 31.5 42
52.5 63
Single conformation possible, 4 copies 10.75 21.5 32.25
43 53.75 64.5
Single conformation possible, 4 copies 11 22 33 44 55
66
Single conformation possible, 4 copies 11.25 22.5 33.75
45 56.25 67.5
Single conformation possible, 4 copies 11.5 23 34.5 46
57.5 69
Single conformation possible, 4 copies 11.75 23.5 35.25
47 58.75 70.5
12 24 36 48 60 72
12.25
24.5 36.75 49 61.25 73.5
12.5 25 37.5 50 62.5
75
Single conformation possible 12.75 25.5 38.25 51
63.75 76.5
Single conformation possible 13 26 39 52 65 78
Cells with heavy dark borders and in bold represent better species, cells with
thin borders and
non-bolded represent species either too small or too large for optimal
packaging. The lower and
higher limits for efficient packaging have been set to 36 kb and 51 kb,
respectively.
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