Note: Descriptions are shown in the official language in which they were submitted.
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
1
BACTERIAL DELIVERY VEHICLES FOR IN VIVO DELIVERY
OF A DNA PAYLOAD
TECHNICAL FIELD
[I] The present disclosure relates generally to bacterial delivery vehicles
and their use in
efficient transfer of a desired payload into a target bacterial cell
population. More
specifically, the present disclosure relates to bacterial delivery vehicles
with desired host
range that can be used to efficiently transfer in vivo the desired payload to
one or more
target bacterial cell populations of the microbiome.
BACKGROUND
[2] Encapsidated DNA in bacterial delivery particles can be used as a method
to deliver
genetic material into a target bacterial population. Several systems exist
that allow the
packaging of exogenous DNA into phage particles, for example bacteriophage
lambda, in
a laboratory setting. Such systems include, for example, a system that
directly produces
the packaged particles in a bacterial cell and in vitro cell-free systems
[1]¨[3]. These
systems exploit the fact that the addition of a cognate packaging site to an
exogenous
DNA vector (called phagemid, and more specifically cosmid in the presence of a
cos
packaging site) allows for the efficient packaging of this payload into a
mature viral
particle. This approach has been used in many different applications, for
example for the
generation of cosmid libraries or the transduction of specific genes into
bacteria [2], [4].
Most of these transduction assays are performed in laboratory conditions:
cells are
cultured in a controlled growth media, such as LB, and the transduction
protocol is
carried out in a buffer with known solute concentrations.
[3] For in vivo applications, such as oral delivery of encapsidated DNA into
particles, the
need exists for bacterial delivery vehicles that can be given at high enough
concentrations
to reach all the target cells; hence, a payload that gives high enough titers
is essential to
optimize the in vivo activity as well as the manufacturing process. The
packaged DNA
into particles also needs to be able to bind its target cell strongly and long
enough for the
injection process to occur.
SUMMARY
[4] Although it was previously shown that the presence of side tail fiber is
not necessary
for lambda-mediated in vitro transduction experiments in K-12 laboratory
strains [5], it is
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
2
demonstrated herein that side tail fiber is actually necessary to optimize in
vivo activity in
a treated subject. The present disclosure provides delivery vehicles for in
vivo delivery to
a target host bacterium of interest, such as those in the gut of a treated
subject.
[5] The present disclosure relates to lambdoid bacterial delivery vehicles and
their use in
efficient in vivo transfer of a desired payload into a target bacterial cell.
The desired
payload includes nucleic acid molecules that encode a gene of interest.
[6] A lambdoid bacterial delivery vehicle for use in in vivo delivery of a DNA
payload of
interest into a targeted bacterial cell population is provided. In one
embodiment, the
bacterial delivery vehicle comprises one or more receptor binding protein(s)
(RBP). 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.
[7] In one embodiment, the bacterial delivery vehicle comprises one or more
RBPs
selected from the group consisting of a functional lambdoid side tail fiber
protein (herein
"STF protein"), a functional lambdoid gpJ protein and a functional lambdoid
gpH protein.
In another embodiment, the bacterial delivery vehicle may comprise two or more
proteins
selected from the group consisting of a functional lambdoid side tail fiber
protein (herein
"STF protein"), a functional lambdoid gpJ protein and a functional lambdoid
gpH
protein. In a particular embodiment, the bacterial delivery vehicle comprises
a functional
lambdoid side tail fiber protein (herein "STF protein") and a functional
lambdoid gpJ
protein. In yet another embodiment, the bacterial delivery vehicle comprises a
functional
lambdoid side tail fiber protein (herein "STF protein"), a functional lambdoid
gpJ protein
and a functional lambdoid gpH protein. In another aspect, the bacterial
delivery vehicle
comprises a functional lambdoid gpJ protein and a functional lambdoid gpH
protein. In
another aspect, the bacterial delivery vehicle comprises (i) a functional
lambdoid side tail
fiber protein. In addition to a functional STF protein, the bacterial delivery
vehicle may
further comprise (ii) a functional lambdoid gpJ protein; and optionally (iii)
a functional
lambdoid gpH protein.
[8] In an embodiment, the STF protein, the gpJ protein and/or the gpH protein
are a wild
type lambda STF, gpJ and/or gpH protein. Alternatively, the STF protein, the
gpJ protein
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
3
and/or the gpH protein are recombinant proteins, preferably non-naturally
occurring
recombinant proteins. In particular, the recombinant STF protein, gpJ protein
and/or gpH
protein may be engineered to target the transfer of the DNA payload of
interest into the
targeted bacterial cell. In a non-limiting example, the recombinant STF
protein may be
engineered to advantageously possess enzymatic activity such as depolymerase
activity
and the target bacterial cell may be an encapsulated bacterial cell. Such
depolymerase
activity is found to increase delivery efficiency, and includes activity
associated with an
endosialidase such as, for example, a KlF endosialidase or activity associated
with a
lyase such as, for example, K5 lyase.
[9] Recombinant STF proteins include, for example, engineered chimeric STF
proteins
and in some instances, the disclosure provides their associated chaperone
(also called
accessory) proteins. Such chaperone proteins assist in the folding of the
chimeric STF
protein. The recombinant engineered chimeric STF protein may comprise a fusion
between a portion of a STF protein derived from a lambdoid bacteriophage,
preferably a
lambda or lambda-like bacteriophage, and a portion of a STF protein derived
from a
corresponding STF protein derived from a different bacteriophage. Such
chimeric STF
protein may comprise a fusion between the N-terminal domain of a STF from a
lambdoid
bacteriophage, preferably a lambda or lambda-like bacteriophage, and the C-
terminal
domain of a different STF. In an embodiment, the chimeric STF protein
comprises or
consists of the amino acid sequence of SEQ ID NO: 14, the amino acid sequence
of SEQ
ID NO: 16, the amino acid sequence of SEQ ID NO: 17, the amino acid sequence
of SEQ
ID NO: 19, the amino acid sequence of SEQ ID NO: 21, the amino acid sequence
of SEQ
ID NO: 44, or the amino acid sequence of SEQ ID NO: 50. In a particular
embodiment,
the chimeric STF protein is STF-V10 (SEQ ID NO: 44). Other examples of
chimeric STF
proteins include STF-V1Of (SEQ ID NO: 45), STF-V10a (SEQ ID NO: 46) and STF-
V10h (SEQ ID NO: 47). The present disclosure also provides synthetic bacterial
delivery
vehicles that are characterized by the presence of an engineered branched
receptor
binding multi-subunit protein complex ("branched-RBP"). The engineered
branched-RBP
comprises two or more associated receptor binding proteins, derived from
bacteriophages,
which associate with one another based on the presence of interaction domains
(IDs). In
a particular embodiment, said engineered branched-RBP comprises two or more
associated STF, derived from bacteriophages, which associate with one another
based on
the presence of 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
4
association of one subunit RBP with another. In specific embodiments the
branched-RBP
may comprise multiple RBP subunits, including, for example, two, three, four,
etc.
subunits. The individual RBP subunit may bring different biological functions
to the
overall engineered branched-RBP. Such functions include but are not limited to
host
recognition and enzymatic activity. Such enzymatic activity includes
depolymerase
activity.
[10] Accordingly, bacterial delivery vehicles are provided which enable
transfer of
a nucleic acid payload, encoding a protein or nucleic acid of interest, into a
desired target
bacterial host cell wherein said bacterial delivery vehicles are characterized
by having a
chimeric STF and/or a branched-RBP as disclosed herein.
[11] Bacterial delivery vehicles are also provided that comprise
recombinant gpJ
proteins. Such gpJ proteins include recombinant gpJ proteins, including
chimeric
proteins, that permit recognition of a bacterial cell receptor other than the
LamB OMP
receptor which is the natural lambda phage receptor on the bacterial cell
surface (14). The
recombinant engineered chimeric gpJ protein may comprise a fusion between a
portion of
a gpJ protein derived from a lambdoid bacteriophage, preferably a lambda or
lambda-like
bacteriophage, and a portion of a gpJ protein derived from a corresponding gpJ
protein
derived from a different bacteriophage. Such chimeric gpJ protein may comprise
a fusion
between the N-terminal domain of a gpJ protein from a lambdoid bacteriophage,
preferably a lambda or lambda-like bacteriophage, and the C-terminal domain of
a
different gpJ protein. In an embodiment, the gpJ protein comprises or consists
of the
amino acid sequence of SEQ ID NO: 10, 11, 12, 13 or 49.
[12] Bacterial delivery vehicles are also provided that 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. The recombinant engineered chimeric gpH protein may comprise a
fusion
between a portion of a gpH protein derived from a lambdoid bacteriophage,
preferably a
lambda or lambda-like bacteriophage, and a portion of a gpH protein derived
from a
corresponding gpH protein derived from a different bacteriophage. Such
chimeric gpH
protein may comprise a fusion between the N-terminal domain of a gpH protein
from a
lambdoid bacteriophage, preferably a lambda or lambda-like bacteriophage, and
the C-
terminal domain of a different gpH protein. In an embodiment, the gpH protein
comprises
or consists of the amino acid sequence of SEQ ID NO: 23 or 24.
[13] In certain aspects, the bacterial delivery vehicles provided herein,
are vehicles
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
wherein the recombinant STF protein, gpJ protein and/or gpH protein are
engineered to
increase the efficiency of transfer of the DNA payload into the targeted
bacterial cell.
Such bacterial cell may be selected from the group consisting of Yersinia
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.,
Listeria spp,
and mixtures thereof, preferably from the group consisting of E.coli. and
other bacterial
species of interest such as, for example, Klebsiella, Citrobacter,
Agrobacterium,
Enterobacter or Pseudomonas, more preferably is E. coli.
[14] The bacterial delivery vehicles disclosed herein provide a means for
transfer,
including in vivo transfer, of a DNA payload of interest into a targeted host
bacterium. In
non-limiting aspects, 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 encoding 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 encoding resistance
to an antibiotic
or to a drug in general, a gene encoding a toxic protein or a toxic factor,
and a gene
encoding a virulence protein or a virulence factor, or any of their
combination. In specific
embodiments, the nucleic acid of interest encodes a therapeutic protein. Still
further, the
nucleic acid of interest may encode an antisense nucleic acid molecule.
[15] In one aspect, the bacterial delivery vehicle enables the transfer of
a nucleic
acid payload that encodes a nuclease that targets cleavage of a host bacterial
cell genome
or a host bacterial cell plasmid. In some aspects, the cleavage occurs in an
antibiotic
resistant gene. In another embodiment, the nuclease mediated cleavage of the
host
bacterial cell genome is designed to stimulate a homologous recombination
event for
insertion of a nucleic acid of interest into the genome of the bacterial cell.
[16] The present disclosure also provides pharmaceutical or veterinary
compositions comprising one or more of the bacterial delivery vehicles
disclosed herein
and a pharmaceutically acceptable carrier. Also provided is a method for
treating a
disease or disorder caused by bacteria, preferably a bacterial infection,
comprising
administering to a subject having a disease or disorder caused by bacteria,
preferably a
bacterial infection, in need of treatment, the provided pharmaceutical or
veterinary
composition. The present disclosure also relates to a pharmaceutical or
veterinary
composition or a bacterial delivery vehicle as disclosed herein for use in the
treatment of
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
6
a disease or disorder caused by bacteria, preferably a bacterial infection. It
further relates
to the use of a pharmaceutical or veterinary composition or a bacterial
delivery vehicle as
disclosed herein for the manufacture of a medicament for treating a disease or
disorder
caused by bacteria, preferably a bacterial infection. The disease or disorder
caused by
bacteria is preferably selected from a bacterial infection, a metabolic
disorder and a
pathology involving bacteria of the human microbiome. More preferably, the
disease or
disorder caused by bacteria is a bacterial infection. A method for reducing
the amount of
virulent and/or antibiotic resistant bacteria in a bacterial population is
provided
comprising contacting the bacterial population with the bacterial delivery
vehicles
disclosed herein. The method may be an in vivo or in vitro method. The present
disclosure
also relates to a pharmaceutical or veterinary composition or a bacterial
delivery vehicle
as disclosed herein for use in reducing the amount of virulent and/or
antibiotic resistant
bacteria in a bacterial population, in particular in a subject having a
bacterial infection. It
further relates to the use of a pharmaceutical or veterinary composition or a
bacterial
delivery vehicle as disclosed herein for the manufacture of a medicament for
reducing the
amount of virulent and/or antibiotic resistant bacteria in a bacterial
population, in
particular in a subject having a bacterial infection.
[17] 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. The methods and compositions described herein
encompass in-
situ bacterial production of any compound of interest, including therapeutic
compounds
such as prophylactic and therapeutic vaccines 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.
BRIEF DESCRIPTION OF FIGURES
[16] In order to better understand the subject matter that is disclosed
herein and to
exemplify how it may be carried out in practice, embodiments will now be
described, by
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
7
way of non-limiting example, with reference to the accompanying drawings. With
specific reference to the drawings, it is stressed that the particulars shown
are by way of
example and for purposes of illustrative discussion of embodiments of the
disclosure.
[17] FIG. 1. Presence of transductants after oral gavage of lambda-PaPa
packaged
psgRNAcos cosmids (DNA payload of SEQ ID NO: 1). Black dots, total number of
MG-
GFP cells. White dots, MG-GFP cells with acquired kanamycin resistance.
[18] FIG. 2. In vivo adaptation of lambda PaPa phages on MG-GFP in the gut
of
mice (n=3). Each line corresponds to an experiment performed in one mouse. X
axis,
days.
[19] FIG. 3 Presence of transductants after oral gavage of Ur-lambda
packaged
pJ23104-GFP cosmids (3 kbp) (DNA payload of SEQ ID NO: 2). Black dots, total
number of MG1655-Str cells. White dots, MG-GFP cells with acquired
chloramphenicol
resistance.
[20] FIG. 4. Presence of transductants after oral gavage of Ur-lambda
packaged
pJF1 cosmids (7 kb) (DNA payload of SEQ ID NO: 3). Black dots, total number of
MG-
GFP cells. White dots, MG-GFP cells with acquired chloramphenicol resistance.
[21] FIG. 5. Titration of packaged lambda phagemids with different payload
sizes.
[22] FIG. 6A-B. Delivery efficiency after oral gavage of Ur-lambda packaged
GG6K and GG8K cosmids (respectively of SEQ ID NO: 6 and SEQ ID NO: 7) with or
without stf. FIG.6A. Packaged phagemids with STF. FIG.6B Packaged phagemids
without STF.
[23] FIG. 7. Alignment of gpJ variants to lambda gpJ. Two insertion points
based
on protein identity, marked with boxes 1 and 2, were chosen to generate
chimeras with
the lambda gpJ.
[24] FIG. 8A-D. Apparent titers of different gpJ chimeras. FIG. 8A. 591
chimeras,
inserted into lambda gpJ using the second insertion point (box #2 in Figure
7). Lambda
WT refers to the original gpJ variant recognizing LamB. Each lane represents a
10-fold
dilution of the produced packaged phagemid, from the most concentrated on the
right to
the most diluted on the left. FIG. 8B. Apparent titers of gpJ variants lambda
WT, Z2145
and 1A2 (respectively of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13) in
three
strains: MG-GFP (black bars), MG-delta-LamB (white bars) and H10-waaJ (0157
strain
lacking the 0157 antigen, grey bars). FIG. 8C. Delivery efficiency (% GFP+
cells)
measured in a flow cytometer of H10 wt strain (contains a group 4 capsule)
using a
lambda packaged phagemid with a gpJ Z2145 variant (SEQ ID NO: 12) or a Z2145
(SEQ
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
8
ID NO: 12) with WW11.2 stf variant (SEQ ID NO: 16). FIG. 8D. Delivery
efficiency
(%GFP+ cells) measured in a flow cytometer with MG1655 or MG1656-OmpC0157
transduced with lambda packaged phagemid comprising the gpJ variants A8 (SEQ
ID
NO: 49) or 1A2 (SEQ ID NO: 13) and a chimeric lambda-P2 STF (SEQ ID NO: 50)
[25] FIG. 9 A-B. Analysis of lambda gpH and generation of engineered
variants.
FIG. 9A. Protein alignment between lambda gpH and a gpH protein from another
lambdoid prophage found in E. coli. FIG. 9B. Titration of lambda WT gpH
variants (left
panels - SEQ ID NO: 23) and engineered gpH-IAI (right panels - SEQ ID NO: 24)
in
MG1655, manZ and manY mutants. Each lane represents a 10-fold dilution of the
produced packaged phagemid, from the most concentrated on the right to the
most diluted
on the left.
[26] FIG. 10. Delivery efficiency of engineered lambda packaged phagemids
in
other Proteobacteria. Dot titrations of different gpJ and STF combinations on
an
Enterobacter cloacae strain. 10 [IL of packaged phagemids were mixed with 90
[IL of
bacteria at an 0D600 -0.7, incubated for 30 min at 37 C and 10 [IL of the
reaction plated
on LB Agar plus 25 1.tg/mL chloramphenicol.
[27] FIG. 11. Stability of 1A2-STF118 or 1A2-5TF29 packaged phagemids in
PBS. Grey 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 [IL.
[28] FIG. 12. 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.
[29] FIG. 13. Relative abundance of Eligobiotics comprising either 3 or 4
copies
of their 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 .
DETAILED DESCRIPTION
[30] The present disclosure relates to bacterial delivery vehicles with
desired host
ranges that can be used to efficiently transfer the desired payload in vivo to
one or more
target bacterial cells of the microbiota of a subject.
[31] Disclosed herein are methods and compositions for in vivo delivery of
a
desired payload into the microbiome of a subject. Such delivery vehicles are
engineered
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
9
to contain as part of their payload, nucleic acids encoding RNA molecules or
proteins that
may be useful for treatment of disorders and diseases of a subject. Such
nucleic acids may
encode generally, any molecules, compounds and proteins, as non-limiting
examples.
[32] 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. 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.
[33] In one aspect, bacterial delivery vehicles with desired target host
ranges are
provided for use in in vivo transfer of a payload to the microbiome of a
subject. The
bacterial delivery vehicle may comprise one or more proteins selected from the
group
consisting of a functional lambdoid side tail fiber protein (herein "STF
protein"), a
functional lambdoid gpJ protein and a functional lambdoid gpH protein. In
another
embodiment, the bacterial delivery vehicle may comprise two or more proteins
selected
from the group consisting of a functional lambdoid side tail fiber protein, a
functional
lambdoid gpJ protein and a functional lambdoid gpH protein. In a particular
embodiment,
the bacterial delivery vehicle comprises a functional lambdoid STF protein and
a
functional lambdoid gpJ protein. In yet another embodiment, the bacterial
delivery
vehicle may comprise a functional lambdoid STF protein, a functional lambdoid
gpJ
protein and a functional lambdoid gpH protein. In another aspect, the
bacterial delivery
vehicle may comprise a functional lambdoid gpJ protein and a functional
lambdoid gpH
protein. In another aspect, the bacterial delivery vehicle may comprise (i) a
functional
lambdoid STF protein. In addition to a functional STF protein, the bacterial
delivery
vehicle may further comprise (ii) a functional lambdoid gpJ protein; and
optionally (iii) a
functional lambdoid gpH protein.
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
[34] In an embodiment, the functional STF protein, the functional gpJ
protein
and/or the functional gpH protein are respectively wild type lambda STF, gpJ
and/or gpH
proteins. Alternatively, the functional STF protein, the functional gpJ
protein and/or the
functional gpH protein are recombinant proteins.
[35] As used herein, the term "recombinant protein" refers to non-naturally
occurring proteins, in particular engineered proteins obtained by
recombination
technique. Such recombinant proteins include, for example, engineered chimeric
proteins.
[36] As used herein, a functional protein means in general a protein with a
biological activity; more specifically a functional wild type, recombinant
protein, variant,
fusion or fragment herein relates to a wild type, recombinant protein,
variant, fusion or
fragment 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.
[37] In the context of the invention, a protein, such as STF, gpJ and gpH
proteins,
may be determined as being functional by titrating packaged phagemids
containing said
protein on bacterial cells known to display receptors recognized by said
protein and
comparing it to the titer obtained with the same packaged phagemids on
bacterial cells
known to display receptors which are not recognized by said protein.
[38] Such recombinant chimeric STF protein may comprise a fusion between a
portion of a STF protein derived from a lambdoid bacteriophage, preferably a
lambda or
lambda-like bacteriophage, and a portion of a STF protein derived from a STF
protein
derived from a different bacteriophage (herein referred to also as a "chimeric
receptor
binding protein" or "chimeric RBP"), in particular from a different lambdoid
bacteriophage or from a non-lambdoid bacteriophage. Such chimeric STF protein
may
comprise a fusion between the N-terminal domain of a STF protein from a
lambdoid
bacteriophage, preferably a lambda or lambda-like bacteriophage, and the C-
terminal
domain of a different STF. As used herein, a receptor binding protein or RBP
may be a
STF derived 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
11
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.
[39] 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 (k). 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 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, gpH and gpJ proteins for use, would be
understood
generally by one skilled in the art.
[40] Lambdoid phages can be defined as belonging to the lambda supercluster
based on genomic analysis [6]. 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 (k), 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), ES18-like (ES18, Oslo, SPN3UB), Gifsy-2-like (gifsy-
2,
gifsy-1, Fels-1, mEp043, mEp213, CP-1639, CTD-I0, mEp640, FSL SP-016), BP-4795-
like (BP-4795, 2851, stx2-1717, YYZ-2008), SW-like (SW, SITI, SfIV, SfI,
01327,
5T64B), P22-like (P22, L, SPN9CC, 5T64T, 5T104, 5T160, epsi1on34, g341, SE1,
Emek, y20, IME10, Sf6, HK620, CUS-3, SPC-P1), APSE-1-like (APSE-1, APSE-2),
933W-like (933W, stx10, stx20-I, stx20-II, stx2-86, min27, 024B, P13374, TL-
2011c,
VT2-sakai, VT20 272), HK639-like (HK639), OES15-like (0E515), H52-like (H52),
ENT47970-like (ENT47670), ZF40-like (ZF40), OEt88-like (0Et88).
[41] In the present disclosure a lambdoid STF protein includes, for
example, a
protein comprising or consisting of an amino acid sequence having at least 75%
identity
up to amino acid corresponding to amino acid 130 of lambda STF (Uniprot P03764
SEQ
ID NO: 14), in particular up to amino acid 130 of said lambda STF; a lambdoid
gpJ
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
12
protein includes, for example, a protein comprising or consisting of an amino
acid
sequence having at least 35% identity up to an amino acid corresponding to
amino acid
606 of lambda gpJ (Uniprot P03749 SEQ ID NO: 10), in particular up to amino
acid 606
of said lambda gpJ; and a lambdoid gpH protein includes, for example, a
protein
comprising or consisting of an amino acid sequence having at least 40%
identity over the
complete length of lambda gpH (Uniprot P03736 SEQ ID NO: 23) and considering
that
the stretch of amino acids between positions 189 and 391 may bear little or no
identity at
all. A lambdoid bacterial delivery vehicle includes a bacterial delivery
vehicle comprising
a functional lambdoid stf protein and/or a functional lambdoid gpJ protein
and/or
functional lambdoid gpH protein, which each may have an altered host range
compared to
the wild-type lambda phage.
[42] In one aspect, the STF protein includes a protein that comprises or
consists of
an amino acid sequence with at least 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: 14, or
with
any of the recombinant STF proteins, fusions, variants or fragments disclosed
herein. In
one aspect, the gpJ protein includes a protein that comprises or consists of
an amino acid
sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity
with the wild
type gpJ protein amino acid sequence of SEQ ID NO: 10, or with any of the
recombinant
gpJ proteins, fusions, variants or fragments disclosed herein. In one aspect,
the gpH
protein includes a protein that comprises or consists of an amino acid
sequence with at
least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the wild type
gpH protein
amino acid sequence of SEQ ID NO: 23, or with any of the recombinant gpH
proteins,
fusions, variants or fragments disclosed herein. In another aspect, nucleic
acids encoding
for such wild type, or recombinant, STF, gpH and gpJ proteins are provided
herein.
[43] 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., % 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 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.
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
13
[44] 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.
[45] A variety of different lambdoid bacterial delivery vehicles are
provided as a
means for transfer of a payload into a target bacterial cell population. Such
bacterial
delivery vehicles include those that comprise one or more wild type lambdoid
STF, gpH
and gpJ proteins. Alternatively, the delivery vehicles may comprise one or
more wild type
STF, gpH, or gpJ proteins combined with one or more recombinant STF, gpH or
gpJ
proteins, including chimeric proteins, fusions, variants or fragments as
disclosed herein.
Included are delivery vehicles wherein the three STF, gpH and gpJ proteins are
wild-type
and those delivery vehicles wherein the three STF, gpH and gpJ proteins are
recombinant,
fusions, variants or fragments.
[46] The present disclosure provides delivery vehicles, for example,
comprising a
chimeric receptor binding protein (RBP), wherein the chimeric RBP comprises a
fusion
between an N-terminal domain of a RBP from a lambdoid bacteriophage,
preferably a
lambda or lambda-like bacteriophage, and a C-terminal domain of a different
bacteriophage RBP. Such bacteriophage RBPs, from which the chimeric RBP are
derived,
include, for example, "L-shape fibers", "side tail fibers (STFs)", "long tail
fibers" or
"tailspikes." Such bacteriophage RBPs, from which the chimeric RBP are
derived, may
be wild-type RBPs or RBP variants, preferably wild-type RBPs. Such chimeric
RBPs
include those having an altered host range and/or biological activity such as,
for example,
depolymerase activity. In an embodiment, the chimeric 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 receptor binding protein (RBP) is
derived from
any bacteriophage or from any bacteriocin.
[47] Such chimeric RBP may comprise a fusion between the N-terminal domain
of
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
14
a RBP from a lambdoid bacteriophage, preferably a lambda or lambda-like
bacteriophage,
and the C-terminal domain of a different RBP. The N-terminal domain is
typically fused
to the N-terminal end of the C-terminal domain.
[48] By "N-terminal domain" of a RBP, in particular of a STF protein, from
a
bacteriophage is meant herein an amino acid region of said RBP starting at the
N-terminal
end of said RBP and ending at positions 80-150, 320-460 or 495-560 of said
RBP, said
positions being with reference to the lambda bacteriophage STF sequence (SEQ
ID NO:
14). By "C-terminal domain" of a RBP, in particular of a STF protein, from a
bacteriophage is meant herein an amino acid region of said RBP starting at
positions 25-
150, 320-460 or 495-560 of said RBP, said positions being with reference to
the lambda
bacteriophage STF sequence (SEQ ID NO: 14), and ending at the C-terminal end
of said
RBP.
[49] In an embodiment, the bacterial delivery vehicles contain a chimeric
RBP
comprising a fusion between an N-terminal domain of a RBP derived from a
lambdoid
bacteriophage, preferably a lambda or lambda-like bacteriophage, and a C-
terminal
domain of a different RBP wherein said N-terminal domain of the chimeric RBP
is fused
to said C-terminal domain of a different RBP within one of the amino acids
regions
selected from positions 80-150, 320-460, or 495-560 of the N-terminal domain
with
reference to the lambda bacteriophage STF sequence (SEQ ID NO: 14). In one
aspect, the
RBP from the lambdoid bacteriophage, preferably the lambda or lambda-like
bacteriophage, and the different RBP 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: 14). 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 RBP with reference to the lambda bacteriophage STF
sequence.
In one specific aspect, the different RBP domain of the chimeric RBP is
derived from a
bacteriophage or a bacteriocin. In one aspect, the chimeric RBP comprises an N-
terminal
domain of a RBP fused to a C-terminal domain of a different RBP within one of
the
amino acids regions selected from positions 80-150, 320-460, or 495-560 of the
N-
terminal RBP domain with reference to the lambda bacteriophage STF sequence
(SEQ ID
NO: 14). In another non-limiting embodiment, the chimeric RBP comprises an N-
terminal domain of a RBP and a C-terminal domain of a different RBP fused
within a site
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
of the N-terminal RBP domain 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: 37), ADAKKS (SEQ ID NO: 38), MDETNR (SEQ ID NO: 39), SASAAA (SEQ ID
NO: 40), and GAGENS (SEQ ID NO: 41).
[50] In a specific embodiment, the chimeric STF protein comprises a fusion
between the N-terminal domain of a lambda bacteriophage STF protein and the C-
terminal domain of a STF protein from another bacteriophage, said N-terminal
domain
being in particular fused to said C-terminal domain within the amino acid
region 495-560
of the N-terminal domain with reference to the lambda bacteriophage STF
protein
sequence (SEQ ID NO: 14). In said embodiment, the chimeric STF variant may be
STF-
V10 comprising or consisting of the amino acid sequence SEQ ID NO: 44 and
typically
encoded by the nucleotide sequence SEQ ID NO: 51. Alternatively, in said
embodiment,
the chimeric STF variant may be WW11.2 comprising or consisting of the amino
acid
sequence SEQ ID NO: 16 and typically encoded by the nucleotide sequence SEQ ID
NO:
30. Still alternatively, in said embodiment, the chimeric STF variant may be
5TF75
comprising or consisting of the amino acid sequence SEQ ID NO: 17 and
typically
encoded by the nucleotide sequence SEQ ID NO: 31. In said embodiment, said
5TF75
may be, in particular be produced, with its associated chaperone protein,
which typically
comprises or consists of the amino acid sequence SEQ ID NO: 18, and is
typically
encoded by the nucleic acid sequence SEQ ID NO: 34. Still alternatively, in
said
embodiment, the chimeric STF variant may be 5TF23 comprising or consisting of
the
amino acid sequence SEQ ID NO: 21 and typically encoded by the nucleotide
sequence
SEQ ID NO: 35. In said embodiment, said 5TF23 may be, in particular be
produced, with
its associated chaperone protein, which typically comprises or consists of the
amino acid
sequence SEQ ID NO: 22 and is typically encoded by the nucleic acid sequence
SEQ ID
NO: 36. In another embodiment, the chimeric STF protein comprises a fusion
between
the N-terminal domain of a lambda bacteriophage STF protein and the C-terminal
domain
of a STF protein from another bacteriophage, said N-terminal domain being in
particular
fused to said C-terminal domain within the amino acid region 320-460 of the N-
terminal
domain with reference to the lambda bacteriophage STF protein sequence (SEQ ID
NO:
14). In said embodiment, the chimeric STF variant may be STF-EB6 comprising or
consisting of the amino acid sequence SEQ ID NO: 19 and typically encoded by
the
nucleotide sequence SEQ ID NO: 33. In said embodiment, said STF-EB6 may be, in
particular be produced, with its associated chaperone protein, which typically
comprises
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
16
or consists of the amino acid sequence SEQ ID NO: 20 and is typically encoded
by the
nucleic acid sequence SEQ ID NO: 32. Alternatively, in said embodiment, the
chimeric
STF variant may be STF lambda-P2 comprising or consisting of the amino acid
sequence
SEQ ID NO: 50, and typically encoded by the nucleotide sequence SEQ ID NO: 56.
In
said embodiment, said STF lambda-P2 may be, in particular be produced, with
its
associated chaperone protein, which typically comprises or consists of the
amino acid
sequence SEQ ID NO: 57 and is typically encoded by the nucleic acid sequence
SEQ ID
NO: 58. Alternatively, the chimeric STF variant may be STF-V10f comprising or
consisting of the amino acid sequence SEQ ID NO: 45, and typically encoded by
the
nucleic acid sequence SEQ ID NO: 52. Alternatively, the chimeric STF variant
may be
STF-V10a comprising or consisting of the amino acid sequence SEQ ID NO: 46,
and
typically encoded by the nucleic acid sequence SEQ ID NO: 53. Alternatively,
the
chimeric STF variant may be STF-V10h comprising or consisting of the amino
acid
sequence SEQ ID NO: 48, and typically encoded by the nucleic acid sequence SEQ
ID
NO: 54.
[51] Recombinant RBP proteins as disclosed herein may need their
associated
chaperone (also called accessory) proteins for proper folding. Such chaperone
proteins
assist in the folding of the chimeric RBP protein. For example, the lambda STF
protein
comprising or consisting of the amino acid sequence SEQ ID NO: 14 needs its
associated
protein, which typically comprises or consists of the amino acid sequence SEQ
ID NO:
15, for proper folding. The need of an associated chaperone protein for proper
folding of
said chimeric RBP protein typically depends on the RBP from which the C-
terminal
region of the chimeric RBP is derived. For example, the chimeric STF protein
5TF75
comprising or consisting of the amino acid sequence SEQ ID NO: 17 needs its
associated
chaperone protein, which typically comprises or consists of the amino acid
sequence SEQ
ID NO: 18, and is typically encoded by the nucleic acid sequence SEQ ID NO:
34, for
proper folding. For example, the chimeric STF protein STF-EB6 comprising or
consisting of the amino acid sequence SEQ ID NO: 19 needs its associated
chaperone
protein, which typically comprises or consists of the amino acid sequence SEQ
ID NO:
20 and is typically encoded by the nucleic acid sequence SEQ ID NO: 32, for
proper
folding. For example, the chimeric STF protein 5TF23 comprising or consisting
of the
amino acid sequence SEQ ID NO: 21 needs its associated chaperone protein,
which
typically comprises or consists of the amino acid sequence SEQ ID NO: 22 and
is
typically encoded by the nucleic acid sequence SEQ ID NO: 36, for proper
folding. For
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
17
example, the chimeric STF protein STF lambda-P2 comprising or consisting of
the amino
acid sequence SEQ ID NO: 50 needs its associated chaperone protein, which
typically
comprises or consists of the amino acid sequence SEQ ID NO: 57 and is
typically
encoded by the nucleic acid sequence SEQ ID NO: 58, for proper folding. Said
chaperone protein may remain attached to said chimeric STF protein after
folding.
Accordingly, in some embodiments, the bacterial delivery vehicles disclosed
herein may
further comprise the chaperone protein associated with the chimeric STF
protein that said
vehicle comprises. Alternatively, said chaperone protein may not remain
attached to said
chimeric STF protein after folding, and may for example be proteolysed, in
particular
auto-proteolysed. Accordingly, in some embodiments, the bacterial delivery
vehicles
disclosed herein do not comprise the chaperone protein associated with the
chimeric STF
protein that said vehicle comprises.
[52] The present disclosure also provides synthetic bacterial delivery
vehicles that
are characterized by the presence of an engineered branched receptor binding
multi-
subunit protein complex ("branched-RBP"). Such delivery vehicles may be used
to
transfer a payload of interest into a bacterial cell of the microbiome. The
engineered
branched-RBP comprises two or more associated receptor binding proteins,
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
branched-RBP
may comprise multiple RBP subunits, including, for example, two, three, four,
etc.
subunits.
[53] The individual RBP subunit may bring different biological functions to
the
overall engineered 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 branched-
RBP
include, but are not limited to, chimeric receptor binding proteins (RBPs)
described
herein that comprise a fusion between the N-terminal domain of a RBP derived
from a
lambdoid bacteriophage, preferably a lambda or lambda-like bacteriophage, and
the C-
terminal domain of a different RBP wherein said chimeric RBP further comprises
an ID
domain.
[54] Accordingly, bacterial delivery vehicles are provided which enable
transfer of
a nucleic acid payload, encoding a protein or nucleic acid of interest, into a
desired target
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
18
bacterial host cell wherein said bacterial delivery vehicles are characterized
by having a
chimeric-RBP or a branched-RBP as disclosed herein. (For chimeric and branched
RBPs
see, US provisional application US 62/802,777, US application 16/696,769 and
US
application 16/726,033, each of which is incorporated by reference in their
entirety).
[55] Bacterial delivery vehicles are also provided that comprise
recombinant gpJ
proteins. Such gpJ proteins include recombinant gpJ proteins, including
chimeric
proteins, 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 249aa conferring capability of
binding to the
LamB receptor [5]. In a particular embodiment, such chimeric gpJ protein may
comprise
a fusion between the N-terminal domain of a gpJ protein from a lambdoid
bacteriophage,
preferably a lambda or lambda-like bacteriophage, and the C-terminal domain of
a
different gpJ protein. The N-terminal domain is typically fused to the N-
terminal end of
the C-terminal domain.
[56] By "N-terminal domain" 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: 10).
By "C-terminal domain" 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: 10), and ending at the C-terminal end of said gpJ
protein.
[57] For production of chimeric gpJ proteins, two insertion points (Figure
7) have
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 of the invention, the
bacterial
delivery vehicles contain a chimeric gpJ protein comprising a fusion between
an N-
terminal domain of a gpJ protein derived from a lambdoid bacteriophage,
preferably a
lambda or lambda-like bacteriophage, and a C-terminal domain of a different
gpJ protein
wherein said N-terminal domain of the chimeric gpJ protein is fused to said C-
terminal
domain of a different gpJ protein within one of the amino acids regions
selected from
positions 810-825, or 950-970 of the N-terminal domain with reference to the
lambda
bacteriophage gpJ protein sequence (SEQ ID NO: 10).
[58] In a specific embodiment, the chimeric gpJ protein comprises a fusion
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
19
between the N-terminal domain of a lambda bacteriophage gpJ protein and the C-
terminal
domain of a gpJ protein from a different bacteriophage, which typically
recognizes and
binds OmpC, said N-terminal domain being in particular fused to said C-
terminal domain
within the amino acid region 950-970 of the N-terminal domain with reference
to the
lambda bacteriophage gpJ protein sequence (SEQ ID NO: 10). In said embodiment,
the
chimeric gpJ variant may be H591 comprising or consisting of the amino acid
sequence
SEQ ID NO: 11 and typically encoded by the nucleotide sequence SEQ ID NO: 25,
said
H591 chimeric gpJ variant typically recognizing and binding OmpC. In another
embodiment, the chimeric gpJ protein comprises a fusion between the N-terminal
domain
of a lambda bacteriophage gpJ protein and the C-terminal domain of a gpJ
protein from a
different bacteriophage, which typically recognizes a receptor present in E.
coli 0157
strains, said N-terminal domain being in particular fused to said C-terminal
domain
within the amino acid region 810-825 of the N-terminal domain with reference
to the
lambda bacteriophage gpJ protein sequence (SEQ ID NO: 10). In said embodiment,
the
chimeric gpJ variant may be Z2145 comprising or consisting of the amino acid
sequence
SEQ ID NO: 12 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 domain of a lambda bacteriophage gpJ protein and the C-terminal
domain of a
gpJ protein from a different bacteriophage, which typically recognizes the
OmpC receptor
present in 0157 strains, said N-terminal domain being in particular fused to
said C-
terminal domain within the amino acid region 950-970 of the N-terminal domain
with
reference to the lambda bacteriophage gpJ protein sequence (SEQ ID NO: 10). In
said
embodiment, the chimeric gpJ variant may be 1A2 comprising or consisting of
the amino
acid sequence SEQ ID NO: 13 and typically encoded by the nucleotide sequence
SEQ ID
NO: 27, said 1A2 chimeric gpJ variant typically recognizing the OmpC receptor
present
in E. coli 0157 strains. In still another embodiment, the chimeric gpJ protein
comprises a
fusion between the N-terminal domain of a lambda bacteriophage gpJ protein and
the C-
terminal domain of a gpJ protein from a different bacteriophage, which
typically
recognizes the OmpC receptor present in both 0157 and MG1655 strains, said N-
terminal
domain being in particular fused to said C-terminal domain within the amino
acid region
950-970 of the N-terminal domain with reference to the lambda bacteriophage
gpJ protein
sequence (SEQ ID NO: 10). In said embodiment, the chimeric gpJ variant may be
A8
comprising or consisting of the amino acid sequence SEQ ID NO: 49 and
typically
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
encoded by the nucleotide sequence SEQ ID NO: 55, said A8 chimeric gpJ variant
typically recognizing the OmpC receptor in both E. coli 0157 and MG1655
strains.
[59] Bacterial delivery vehicles are also provided that 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. The recombinant engineered chimeric gpH protein may comprise a
fusion
between a portion of a gpH protein derived from a lambdoid bacteriophage,
preferably a
lambda or lambda-like bacteriophage, and a portion of a gpH protein derived
from a
corresponding gpH protein derived from a different bacteriophage. Such
chimeric gpH
protein may comprise a fusion between the N-terminal domain of a gpH protein
from a
lambdoid bacteriophage, preferably a lambda or lambda-like bacteriophage, and
the C-
terminal domain of a different gpH protein. One such variant is gpH-IAI of
amino acid
sequence SEQ ID NO: 24 and nucleotide sequence SEQ ID NO: 28.
[60] In a particular embodiment, said bacterial delivery vehicle comprises
chimeric
STF-V10h variant as disclosed above and chimeric 1A2 variant as disclosed
above.
[61] In aspects, the bacterial delivery vehicles provided herein, are
vehicles
comprising recombinant STF protein(s) (including but not limited to chimeric
and branched
RBPs), gpJ protein(s) and/or gpH protein(s) that are 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.coli. and other bacterial species of
interest.
[62] It has also been demonstrated herein that the size of the packaged
genome can
have effects on the efficiency of packaging.
[63] Nucleic acid molecules encoding the wild type, as well as recombinant
STF,
gpJ, and gpH proteins disclosed herein are provided. Such nucleic acids may be
included
in vectors such as bacteriophages, plasmids, phagemids, viruses, and other
vehicles which
enable transfer and expression of the recombinant STF, gpJ, and gpH encoding
nucleic
acids.
[64] In a particular embodiment, nucleic acids are included in a single
vector. In a
more particular embodiment, said vector comprises or consists of the nucleic
acid
sequence SEQ ID NO: 47.
[65] 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. 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
21
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.
[66] 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. 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 wild-type and recombinant STF, gpJ and gpH
proteins.
[67] The present disclosure also provides a production cell line producing
the
bacterial delivery vehicles disclosed herein.
[68] 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
22
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, like an recombinant 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. In a particular embodiment, said
production cell
line produces:
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 14 and its associated chaperone comprising or consisting of the amino acid
sequence
of SEQ ID NO: 15,
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 16,
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 17 and its associated chaperone comprising or consisting of the amino acid
sequence
of SEQ ID NO: 18,
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 19 and its associated chaperone comprising or consisting of the amino acid
sequence
of SEQ ID NO: 20,
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 21 and its associated chaperone comprising or consisting of the amino acid
sequence
of SEQ ID NO: 22,
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 44, or
- a STF protein which comprises or consists of the amino acid sequence of
SEQ ID
NO: 50 and optionally its associated chaperone comprising or consisting of the
amino
acid sequence of SEQ ID NO: 57.
[69] In a particular embodiment, said helper phage comprises a nucleic
acid
sequence encoding the chimeric RBP comprising or consisting of the sequence
SEQ ID
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
23
NO: 48, said nucleic acid sequence typically comprising or consisting of the
sequence
SEQ ID NO: 54, and said helper phase optionally further comprises a nucleic
acid
sequence encoding the chimeric gpJ variant comprising or consisting of the
sequence
SEQ ID NO: 13, said nucleic acid sequence typically comprising or consisting
of the
sequence SEQ ID NO: 27. In a particular embodiment, said helper phage is a
lambda
phage 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: 48, said nucleic acid sequence typically comprising or
consisting
of the sequence SEQ ID NO: 54, (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: 13, said nucleic acid
sequence
typically comprising or consisting of the sequence SEQ ID NO: 27, 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 cI repressor, such
as the cI857
version.
[70] In an embodiment, the bacterial delivery vehicle disclosed herein
comprises a
DNA payload of interest. 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.
[71] As shown in Example 1 below, the efficiency of loading of the payload
by the
bacterial delivery vehicle disclosed herein may depend upon the size of the
payload,
among others. Accordingly, in a particular embodiment, the payload has a size
superior or
equal to 4 kb, and preferably inferior or equal to 51 kb.
[72] 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 .
[73] The inventors more particularly demonstrated that it was possible to
produce a
more uniform population of bacterial delivery vehicles comprising an almost
unique
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
24
number of payload copies when said payload had a size of a specific range.
[74] 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.
[75] 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.
[76] 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.
[77] 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 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.
[78] Origins of replication known in the art have been identified from
species-
specific plasmid DNAs (e.g. CoIE1, R1, pT181, pSC101, pMB1, R6K, RK2, pl5a and
the
like), from bacterial virus (e.g. yX174, M13, Fl and P4) and from bacterial
chromosomal
origins of replication (e.g. oriC). In one embodiment, the phagemid according
to the
disclosure comprises a bacterial origin of replication that is functional in
the targeted
bacteria.
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
[79] 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.
[80] 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.
[81] 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.
[82] 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).
[83] 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.
[84] Origins of replication used on the plasmid of the disclosure may be of
moderate copy number, such as colE1 ori 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).
[85] In one embodiment, the bacterial origin of replication is selected in
the group
consisting of ColE1, pMB1 and variants (pBR322, pET, pUC, etc), pl5a, ColA,
ColE2,
pOSAK, pSC101, R6K, IncW (pSa etc), IncFII, pT181, Pl, F IncP, IncC, IncJ,
IncN,
IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5, pPS10, pC194,
pE194,
BBR1, pBC1, pEP2, pWV01, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4,
0E01, 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.
[86] In an embodiment, the bacterial origin of replication is a E.coli
origin of
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
26
replication selected in the group consisting of ColE1, pMB1 and variants
(pBR322, pET,
pUC, etc), pl5a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (pSa etc), IncFII,
pT181,
Pl, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13,
NTP16,
R1, f5 and pPS10.
[87] In an embodiment, the bacterial origin of replication is selected in
the group
consisting of pC194, pE194, BBR1, pBC1, pEP2, pWV01, pLF1311, pAP1, pWKS1,
pLS1, pLS11, pUB6060, pJD4, pll101, 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.
[88] In an embodiment, the bacterial origin of replication is ColEl.
[89] 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.
[90] 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.
[91] In an embodiment, the phage origin of replication can be the wild-type
or non-
wild type sequence of the M13, fl, yX174, P4, lambda, P2, lambda-like, HK022,
mEP237, HK97, HK629, HK630, mEP043, mEP213, mEP234, mEP390, mEP460,
mEPxl, mEPx2, phi80, mEP234, T2, T4, T5, T7, RB49, phiX174, R17, PRD1 P1-like,
P2-like, P22, P22-like, N15 and N15-like bacteriophages.
[92] In an embodiment, the phage origin of replication is selected in the
group
consisting of phage origins of replication of M13, fl, yX174, P4, and lambda.
[93] In a particular embodiment, the phage origin of replication is the
lambda or P4
origin of replication.
[94] The delivered nucleic acid of interest 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 encoding 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 encoding resistance
to an antibiotic
or to a drug in general, a gene encoding a toxic protein or a toxic factor,
and a gene
encoding a virulence protein or a virulence factor, or any of their
combination. In an
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
27
embodiment, the nucleic acid payload encodes a therapeutic protein. In another
embodiment, the nucleic acid payload encodes an antisense nucleic acid
molecule.
[95] 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 II 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).
[96] 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.
[97] 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 interest may comprise proteins and enzymes achieving a useful
function such
as modifying the metabolism of the bacteria or the composition of its
environment.
[98] In a particular embodiment, the nucleic sequence of interest is
selected in the
group consisting of Cas9, a single guide RNA (sgRNA), a CRISPR locus, a gene
encoding 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 encoding resistance to an antibiotic or to a drug in general,
a gene
encoding a toxic protein or a toxic factor and a gene encoding a virulence
protein or a
virulence factor.
[99] In a particular embodiment, the delivered nucleic acid sequence
according to
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
28
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).
[100] 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.
[101] 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.
[102] 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 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 I, III and IV. Class 2 is made of single-unit effector modules, like Cas9
nuclease, and
includes type II (II-A,II-B,II-C,II-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,VI-B1,VI-B2,VI-C,VI-D)
[103] The sequence of interest according to the present disclosure
comprises a
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
29
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 II 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 encoding resistance to a drug in general.
[104] Non-limiting examples of Cas proteins as part of a multi-subunit
effector or as
a single-unit effector include Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6,
Cas7, Cas8,
Cas9 (also known as Csnl and Csx12), Cas10, Cash1 (SS), Cas12a (Cpfl), Cas12b
(C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10,
C2c9, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Cscl, Csc2,
Csel,
Cse2, Csyl, Csy2, Csy3, Csfl, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6,
Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10,
Csx16,
CsaX, Csx13, Csxl, Csx15, SdCpfl, CmtCpfl, TsCpfl, CmaCpfl, PcCpfl, ErCpfl,
FbCpfl, UbcCpfl, AsCpfl, LbCpfl, 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.
[105] 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.
[106] By "Cas9" is meant a protein Cas9 (also called Csnl 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).
[107] 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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).
[108] The sequence encoding Cpfl (Cas12a) (the entire protein or a fragment
thereof) as used in the context of the disclosure can be obtained from any
known Cpfl
(Cas12a) protein (Koonin et al., 2017). Examples of Cpfl(Cas12a) proteins
useful in the
present disclosure include, but are not limited to, Cpfl(Cas12a) proteins of
Acidaminococcus sp, Lachnospiraceae bacteriu and Francisella novicida.
[109] 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).
[110] 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.
[111] In a particular embodiment, the nucleic sequence of interest is 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 encoding a bacterial receptor, a membrane protein, a
structural
protein, a secreted protein, and a gene encoding resistance to a drug in
general.
[112] 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. 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, Stxl
(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, cnfl, hra, sat, ireA, usp ompT,
ibeA, malX,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
31
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, stxl and stx2.
For example,
such targeted virulence factor gene can be Yersinia pestis virulence factor
gene such as,
without limitation, yscF (plasmid-borne (pCD1) T3SS external needle subunit).
For
example, such targeted virulence factor gene can be Francisella tularensis
virulence
factor gene such as, without limitation, fs1A. 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 ctxB (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 baumannii virulence 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 to1C. 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.
[113] In another embodiment, the CRISPR/Cas9 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, SHY, TEM, BlaB, CcrA, IMP, NDM, VIM,
ACT, AmpC, CMY, LAT, PDC, OXA 13-lactamase, mecA, 0mp36, OmpF, PIB, bla
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
32
(blaI, blaR1) and mec (mecI, mecR1) operons, Chloramphenicol acetyltransferase
(CAT), Chloramphenicol phosphotransferase, Ethambutol-resistant
arabinosyltransferase
(EmbB), MupA, MupB, Integral membrane protein MprF, Cfr 23S rRNA
methyltransferase, Rifampin ADP-ribosyltransferase (An), Rifampin
glycosyltransferase,
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 acetyltransferase,
Fluoroquinolone
acetyltransferase, 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, Macrofide
phosphotransferases (MPH), MefA, MefE, Mel, Streptothricin acetyltransferase
(sat),
Sull, Sul2, Sul3, sulfonamide-resistant FolP, Tetracycline inactivation enzyme
TetX,
TetA, TetB, TetC, Tet30, Tet31, TetM, Tet0, TetQ, Tet32, Tet36, MacAB-To1C,
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/).
[114] In another embodiment, the CRISPR/Cas9 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. coli ST), colibactin, BFT (B. fragilis
toxin) or any toxin
described in Henkel et al., (Toxins from Bacteria in EXS. 2010; 100: 1-29).
[115] In a particular embodiment, said payload comprises or consists of the
nucleic
acid sequence SEQ ID NO: 47.
[116] 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 and/or the branched-RBPs expressed by
the
delivery vehicles with the bacterial cell. It can be any commensal, symbiotic
or
pathogenic bacteria of the microbiota or microbiome.
[117] A microbiome may comprise a variety of endogenous bacterial species,
any of
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
33
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.
[118] 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., Brucella 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.
[119] 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.
[120] In an embodiment, the targeted bacteria can be selected from the
group
consisting of Yersinia 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.
[121] 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
coli, Shewanella oneidensis and Listeria. Anaerobic bacterial cells also
include obligate
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
34
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. B acteriophages 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.
[122] 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
actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori,
Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma
mycoides,
Treponema denticola, Bacillus thuringiensis, Staphilococcus 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 fragilis,
Staphylococcus
epidermidis, Streptomyces phaechromo genes, Streptomyces ghanaenis, Klebsiella
pneumoniae, Enterobacter cloacae, Enterobacter aero genes, Serratia
marcescens,
Morganella morganii, Citrobacter freundii, Propionibacterium freudenreichii,
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 coli, Camp ylobacter fetus, Chlamydia
pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium
botulinum,
Clostridium difficile, Clostridium perfringens, Clostridium tetani,
Corynebacterium
diphtheria, Cutibacterium acnes (formerly Propionibacterium acnes), Ehrlichia
canis,
Ehrlichia chaffeensis, Enterococcus faecium, Francisella tularensis,
Haemophilus
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
influenza, Legionella pneumophila, Leptospira interrogans, Leptospira
santarosai,
Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, 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 pyo genes, Gardnerella vaginalis, 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 coli,
Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter
baumanii, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter aero
genes,
and a mixture thereof.
[123] 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, Weisella, Clostridium,
Bacteroides,
Ruminococcus, Faecalibacterium, Treponema, Phascolarctobacterium, Megasphaera,
Faecalibacterium, Bifidobacterium, Lactobacillus, Sutterella, and/or
Prevotella.
[124] In other embodiments, the targeted bacteria cells are, without
limitation,
Achromobacter xylosoxidans, Acidaminococcus fermentans, Acidaminococcus
intestini,
Acidaminococcus sp., Acinetobacter baumannii, Acinetobacter junii,
Acinetobacter
lwoffii, Actinobacillus capsulatus, Actinomyces naeslundii, Actinomyces neuii,
Actinomyces odontolyticus, Actinomyces radingae, Adlercreutzia equolifaciens,
Aeromicrobium mass iliense, Aggregatibacter actinomycetemcomitans, Akkermansia
muciniphila, Aliagarivorans marinus, Alistipes fine goldii, Alistipes
indistinctus, Alistipes
mops, Alistipes onderdonkii, Alistipes put redinis, Alistipes senegalensis,
Alistipes shahii,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
36
Alistipes timonensis, Alloscardovia omnicolens, Anaerobacter polyendosporus,
Anaerobaculum hydrogeniformans, Anaerococcus hydrogenalis, Anaerococcus
prevotii,
Anaerococcus senegalensis, Anaerofustis stercorihominis, Anaerostipes caccae,
Anaerostipes hadrus, Anaerotruncus colihominis, Aneurinibacillus
aneurinilyticus,
Bacillus licheniformis, Bacillus mass ilioanorexius, Bacillus
massiliosenegalensis,
Bacillus simplex, Bacillus smithii, Bacillus subtilis, Bacillus thuringiensis,
Bacillus
timonensis, Bacteriodes xylanisolvens, Bacteroides acidifaciens, Bacteroides
caccae,
Bacteroides capillosus, Bacteroides cellulosilyticus, Bacteroides clarus,
Bacteroides
coprocola, Bacteroides coprophilus, Bacteroides dorei, Bacteroides eggerthii,
Bacteroides faecis, Bacteroides fine goldii, 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, Bacteroidespectinophilus ATCC, Barnes iella intestinihominis,
Bavariicoccus seileri, Bifidobacterium adolescentis, Bifidobacterium
angulatum,
Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve,
Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium
gallicum,
Bifidobacterium longum, 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 formatexigens, 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
37
Coprobacter fastidiosus, Coprococcus catus, Coprococcus comes, Coprococcus
eutactus,
Corynebacterium ammonia genes, Corynebacterium amycolatum, Corynebacterium
pseudodiphtheriticum, Cutibacterium acnes, Dermabacter hominis,
Desulfitobacterium
hafiliense, Desulfovibrio fairfieldensis, Desulfovibrio piger, Dialister
succinatiphilus,
Dielma fastidiosa, Dorea formicigenerans, Dorea longicatena, Dysgonomonas
capnocytophagoides, Dysgonomonas gadei, Dysgonomonas moss ii, Edwardsiella
tarda,
Eggerthella lenta, Eisenbergiella tayi, Enorma massiliensis, Enterobacter aero
genes,
Enterobacter asburiae, Enterobacter cancero genus, Enterobacter cloacae,
Enterobacter
massiliensis, Enterococcus casseliflavus, Enterococcus durans, Enterococcus
faecalis,
Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum,
Enterococcus
sp., Enterovibrio nigri cans, Erysipelatoclostridium ramosum, Escherichia
coli,
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 necrophorum, Fusobacterium nucleatum, Fusobacterium
periodonticum, Fusobacterium sp., Fusobacterium ulcerans, Fusobacterium
varium,
Gallibacterium anatis, Gemmiger formicilis, Gordonibacter pamelaeae, Hafilia
alvei,
Helicobacter bilis, Helicobacter bills, Helicobacter canadensis, Helicobacter
canis,
Helicobacter cinaedi, Helicobacter macacae, Helicobacter pametensis,
Helicobacter
pullorum, Helicobacter pylori, Helicobacter rodentium, Helicobacter win
ghamensis,
Herbaspirillum massiliense, Holdemanella biformis, Holdemania fdiformis,
Holdemania
filiformis, Holdemania massiliensis, Holdemaniafiliformis, Hungatella
hathewayi,
Intestinibacter bartlettii, Intestinimonas butyriciproducens, Klebsiella
oxytoca, Klebsiella
pneumoniae, Kurthia massiliensis, Lachnospira pectinoschiza, Lactobacillus
acidophilus,
Lactobacillus amylolyticus, Lactobacillus animalis, 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
vaginalis,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
38
Lactobacillusplantarum subsp., Leuconostoc mesenteroides, Leuconostoc
pseudomesenteroides, Listeria grayi, Listeria innocua, Mannheimia
granulomatis,
Marvinbryantia formatexigens, Megamonas ftiniformis, Megamonas hyperme gale,
Methanobrevibacter smithii, Methanobrevibacter smithiiF1, Micrococcus luteus,
Microvirgula aerodenitrifi cans, Mitsuokella jalaludinii, Mitsuokella
multacida,
Mollicutes bacterium, Murimonas intestini, Neisseria macacae, Nitriliruptor
alkaliphilus,
Oceanobacillus mass iliensis, Odoribacter laneus, Odoribacter splanchnicus,
Ornithobacterium 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, Prevotella stercorea, Prevotella
veroralis,
Propionibacterium acnes, Propionibacterium avidum, Propionibacterium
freudenreichii,
Propionimicrobium lymphophilum, Proteus mirabilis, Proteuspenneri ATCC,
Providencia alcalifaciens, Providencia rettgeri, Providencia rustigianii,
Providencia
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,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
39
Streptococcus infantis, Streptococcus lutetiensis, Streptococcus merionis,
Streptococcus
mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus ovis,
Streptococcus
parasanguinis, Streptococcus plurextorum, Streptococcus porci, Streptococcus
pyo genes,
Streptococcus salivarius, Streptococcus sobrinus, Streptococcus the rmophilus,
Streptococcus thoraltensis, Streptomyces albus, Subdoligranulum variabile,
Succinatimonas hippei, Sutterella parvirubra, Sutterella wadsworthensis,
Terrisporobacter glycolicus, Terrisporobacter mayombei, Thalassobacillus
devorans,
Timonella senegalensis, Turicibacter sanguinis, unknown sp, unknown sp.,
Varibaculum
cambriense, Veillonella atypica, Veillonella dispar, Veillonella parvula,
Vibrio
cincinnatiensis, Virgibacillus salexigens and Weissella paramesenteroides.
[125] 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 denitrifi cans, 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 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
Burkholderia terrestris, Burkholderia udeis, Buttiauxella gaviniae, Caenimonas
terrae,
Capnocytophaga gin givalis, Chitinophaga dinghuensis, 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, Ens ifer adhaerens, Ens ifer
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 son gnenensis, 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, Micromonospora citrea, Micromonospora coxensis, Micromonospora
echinofusca, Micromonospora halophytica, Micromonospora kangleipakensis,
Micromonospora maritima, Micromonospora nigra, Micromonospora
purpureochromo gene, Micromonospora rhizosphaerae, Micromonospora
saelicesensis,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
41
Microvirga subterranea, Microvirga 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
llatzerense, Mycobacterium neoaurum, Mycobacterium neworleansense,
Mycobacterium
obuense, Mycobacterium peregrinum, Mycobacterium saopaulense, Mycobacterium
septicum, Mycobacterium setense, Mycobacterium smegmatis, Neisseria subflava,
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 tern cola, Paraburkholderia xenovorans, Paracoccus
laeviglucosivorans, Patulibacter ginsengiterrae, Polymorphospora rubra,
Porphyrobacter colymbi, Prevotella jejuni, Prevotella melaninogenica,
Propionibacterium acnes subsp. elongatum, Proteus vulgaris, Providencia
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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
42
texasensis, Rheinheimera tilapiae, Rhizobium alamii, Rhizobium azibense,
Rhizobium
binae, Rhizobium daejeonense, Rhizobium endophyticum, Rhizobium etli,
Rhizobium
fabae, Rhizobium freirei, Rhizobium gallicum, Rhizobium loessense, Rhizobium
sophoriradicis, Rhizobium taibaishanense, Rhizobium vallis, 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 fttliginis,
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. carnaticus, Streptomyces caeruleatus, Streptomyces canarius,
Streptomyces
capoamus, Streptomyces ciscaucasicus, Streptomyces griseorubiginosus,
Streptomyces
olivaceoviridis, Streptomyces panaciradicis, Streptomyces phaeopurpureus,
Streptomyces
pseudovenezuelae, 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
card iffensis, 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
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
43
mass iliense, 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 vaginalis,
Aquabacterium
citratiphilum, Aquabacterium olei, Aquabacterium olei, Aquabacterium parvum,
Aquincola tertiaricarbonis, Arcobacter venerupis, Arsenicicoccus bolidensis,
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 valvarum, 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,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
44
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 tuberculostearicum, Corynebacterium
tuberculostearicum, Corynebacterium ureicelerivorans, Corynebacterium
variabile,
Couchioplanes caeruleus subsp. caeruleus, Cupriavidus metallidurans,
Curtobacterium
herbarum, Dechloromonas agitata, Deinococcus actinosclerus, Deinococcus
antarcticus,
Deinococcus caeni, Deinococcus ficus, Deinococcus geothermalis, Deinococcus
radiodurans, Deinococcus wulumuqiensis, Deinococcus xinfiangensis, Dermabacter
hominis, Dermabacter vaginalis, Dermacoccus nishinomiyaensis, Desemzia
incerta,
Desertibacter roseus, Dialister invisus, Dialister micraerophilus, Dialister
propionicifaciens, Dietzia aurantiaca, 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,
Fine goldia 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
finhuaensis,
Gordonia kroppenstedtii, Gordonia polyisoprenivorans, Gordonia
polyisoprenivorans,
Granulicatella adiacens, Granulicatella elegans, Haemophilus parainfluenzae,
Haemophilus sputorum, Halomonas sulfidaeris, Herpetosiphon aurantiacus,
Hydrocarboniphaga effusa, Idiomarina mar, Janibacter anophelis, Janibacter
hoylei,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
Janibacter indicus, Janibacter limosus, Janibacter melonis, Jeotgalicoccus
halophilus,
Jon quetella anthropi, Kaistia geumhonensis, Kin gella denitrifi cans, Kin
gella 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 tern cola, Lysinibacillus ftisiformis, Lysobacter
spongiicola,
Lysobacter xinjiangensis, Macrococcus caseolyticus, Marmori cola pocheonensis,
Marmori cola 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 pet rolearium,
Microbacterium phyllosphaerae, Microbacterium resistens, Micrococcus
antarcticus,
Micrococcus cohnii, Micrococcus flavus, Micrococcus lylae, Micrococcus
terreus,
Microlunatus aurantiacus, Micropruina glycogenica, Microvirga aerilata,
Microvirga
aerilata, Microvirga subterranea, Microvirga vignae, Microvirga zambiensis,
Microvirgula aerodenitrifi cans, 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
46
sediminis, Nosocomiicoccus ampullae, Noviherbaspirillum malthae,
Novosphingobium
lindaniclasticum, Novosphingobium rosa, Ochrobactrum rhizosphaerae, Olsenella
uli,
Ornithinimicrobium murale, Omithinimicrobium tianjinense, Oryzobacter terrae,
Ottowia 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 gin givalis, Porphyromonas gin givicanis,
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
pallens, 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, 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
47
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, Rube llimicrobium roseum, Rubrobacter
bracarensis, Rudaea cellulosilytica, Ruminococcus gnavus, Runella zeae,
Saccharopolyspora rectivirgula, Salinicoccus qingdaonensis, Scardovia wig
gsiae,
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 the
rmophilus,
Tianweitania sediminis, Tianweitania sediminis, Treponema amylovorum,
Treponema
denticola, Treponema lecithinolyticum, Treponema medium, Turicella otitidis,
Turicibacter sanguinis, Undibacterium oligocarboniphilum, Undibacterium
squillarum,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
48
Vagococcus salmoninarum, Varibaculum cambriense, Vibrio metschnikovii,
Xanthobacter tagetidis, Xenophilus aerolatus, Xenophilus arseniciresistens,
Yimella
lutea, Zimmermannella alba, Zimmermannella bifida and Zoo gloea caeni.
[126] 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 mass iliense, 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
mass iliensis, Agrobacterium tumefaciens, Algoriphagus aquatilis, Aliivibrio
wodanis,
Alistipes fine goldii, Alloiococcus otitis, Alloprevotella tannerae,
Alloscardovia
omnicolens, Altererythrobacter epoxidivorans, Ammoniphilus oxalaticus,
Amnibacterium
kyonggiense, Anaerococcus hydrogenalis, Anaerococcus lactolyticus,
Anaerococcus
murdochii, Anaerococcus obesiensis, Anaerococcus prevotii, Anaerococcus tet
radius,
Anaerococcus vaginalis, 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 zoo gleoformans, Barnes iella visceri
cola,
Bhargavaea cecembensis, Bifidobacterium adolescentis, Bifidobacterium bifidum,
Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium logum subsp.
infantis,
Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium
scardovii,
Bilophila wadsworthia, Blautia hydrogenotrophica, Blautia obeum, Blautia
producta,
Brachybacterium faecium, Bradyrhizobium japonicum, Brevibacterium mcbrellneri,
Brevibacterium otitidis, Brevibacterium paucivorans, Bulleidia extructa,
Burkholderia
ftingorum, Burkholderia phenoliruptix, Caldicellulosiruptor saccharolyticus,
Caldimonas
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
49
taiwanensis, Campylobacter gracilis, Campylobacter hominis, Campylobacter
sputorum,
Campylobacter ureolyticus, Capnocytophaga ochracea, Cardiobacterium hominis,
Catonella morbi, Chlamydia trachomatis, Chlamydophila abortus, Chondromyces
robustus, Chryseobacterium aquaticum, 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 sin gulare, 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 coli,
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 vaginalis,
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, Hafilia 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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 vaginalis, Lactococcus
lactis,
Leptotrichia buccalis, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc
garlicum, Leuconostoc lactis, Leuconostoc mesentero ides, Lysinimonas
kribbensis,
Mageeibacillus indolicus, Maribacter orientalis, Marinomonas protea,
Marinospirillum
insulare, Massilia timonae, Megasphaera elsdenii, Megasphaera micronuciformis,
Mesorhizobium amorphae, Methylobacterium radiotolerans, Methylotenera
versatilis,
Microbacterium halophilum, Micrococcus luteus, Microterri cola viridarii,
Mobiluncus
curtisii, Mobiluncus mulieris, Mogibacterium timidum, Moorella 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 subflava, Nevskia ramosa, Nevskia
soli,
Nitriliruptor alkaliphilus, Odoribacter splanchnicus, Oligella urethralis,
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 carotovo rum, Pediococcus acidilactici, Pediococcus
ethanolidurans,
Pedobacter alluvionis, Pedobacter wanjuense, Pelomonas aquatica, Peptococcus
niger,
Peptoniphilus asaccharolyticus, Peptoniphilus gorbachii, Peptoniphilus harei,
Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus mass
iliensis,
Peptostreptococcus anaerobius, Peptostreptococcus massiliae,
Peptostreptococcus
stomatis, Photobacterium angustum, Photobacterium frigidiphilum,
Photobacterium
phosphoreum, Porphyromonas asaccharolytica, Porphyromonas bennonis,
Porphyromonas catoniae, Porphyromonas endodontalis, Porphyromonas gin givalis,
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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
51
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 ftilva, 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, San guibacter keddieii, Sediminibacterium salmoneum, Selenomonas
bovis,
Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Shewanella
algae,
Shewanella amazonensis, Shigella boydii, Shigella sonnei, Slackia exigua,
Sneathia
amnii, Sneathia sanguinegens, Solobacterium moo rei, Sorangium cellulosum,
Sphingobium amiense, Sphingobium japonicum, Sphingobium yanoikuyae,
Sphingomonas
wittichii, Sporosarcina aquimarina, Staphylococcus aureus, Staphylococcus
auricularis,
Staphylococcus cap itis, Staphylococcus epidermidis, Staphylococcus
haemolyticus,
Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus
saprophyticus,
Staphylococcus schleiferi, Staphylococcus simiae, Staphylococcus simulans,
Staphylococcus warneri, 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
salivarius,
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, Veillonella atypica, Veillonella
dispar,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
52
Veillonella montpellierensis, Veillonella parvula, Virgibacillus proomii,
Viridibacillus
arenosi, Viridibacillus arvi, Weissella cibaria, Weissella soli, Xanthomonas
campestris,
Xanthomonas vesicatoria, Zobellia laminariae and Zoo gloea ramigera.
[127] In one embodiment, the targeted bacteria are Escherichia coli. In a
particular
embodiment, said targeted bacteria are Shiga-Toxin producing E. coli (STEC).
[128] The targeted bacterial cell population may comprise one or several
bacteria of
interest as defined above. In particular, the targeted bacterial cell
population may
comprise Escherichia coli and one or several other bacteria of interest as
defined above.
[129] 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.
[130] In one embodiment, the targeted bacteria are pathogenic bacteria. The
targeted
bacteria can be virulent bacteria.
[131] The targeted bacteria can be antibacterial resistance bacteria,
including those
selected from the group consisting of extended-spectrum beta-lactamase-
producing
(ESBL) Escherichia coli, ESBL Klebsiella pneumoniae, vancomycin-resistant
Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA),
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.
[132] Alternatively, the targeted bacterium can be a bacterium of the
microbiome of
a given species, including a bacterium of the human microbiota.
[133] The present disclosure is directed to a bacterial delivery vehicle
containing the
payload as described herein. The bacterial delivery vehicles are prepared from
bacterial
virus. The bacterial delivery vehicles are chosen in order to be able to
introduce the
payload into the targeted bacteria.
[134] 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.
[135] Bacteriophages may be selected from the family Myoviridae (such as,
without
limitation, genus Cp220virus, Cp8virus, Ea214virus, Felixolvirus, Mooglevirus,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
53
Suspvirus, Hp lvirus, P2virus, Kayvirus, P10 virus, Silviavirus, Spolvirus,
Tsarbombavirus, Twortvirus, Cc31virus, Jd18virus, Js98virus, Kp15virus,
Moonvirus,
Rb49virus, Rb69virus, S16virus, Schizot4virus, Sp18virus, T4virus, Cr3virus,
Selvirus,
V5virus, Abouovirus, Agatevirus, Agrican357virus, Ap22virus, Arvlvirus,
B4virus,
Bastillevirus, Bc43 lvirus, Bcep78virus, Bcepmuvirus, Biquartavirus,
Bxzlvirus,
Cd119virus, Cp5lvirus, CvmlOvirus, Eah2virus, Elvirus, Hapunavirus,
Jimmervirus,
KpplOvirus, Ml2virus, Machinavirus, Marthavirus, Msw3virus, Muvirus,
Myohalovirus,
Nitivirus, P1 virus, Pakpunavirus, Pbunavirus, Phikzvirus, Rheph4virus,
Rs12virus,
Rslunavirus, Secunda5virus, Seplvirus, Spn3virus, Svunavirus, Tglvirus,
Vhmlvirus and
Wphvirus).
[136] Bacteriophages may be selected from the family Podoviridae (such as,
without
limitation, genus Frilvirus, Kp32virus, Kp34virus, Phikmvvirus, Pradovirus,
Sp6virus,
T7virus, Cplvirus, P68virus, Phi29virus, Nona33virus, Pocjvirus, T12011virus,
Bcep22virus, Bpplvirus, Cba4 lvirus, Dfll2virus, Ea92virus, Epsilonl5virus,
F116virus,
G7cvirus, Jwalphavirus, Kflvirus, Kpp25virus, Litivirus, Luz24virus,
Luz7virus,
N4virus, Nonanavirus, P22virus, Pagevirus, Phieco32virus, Prtbvirus,
Sp58virus,
Una961virus and Vp5virus).
[137] Bacteriophages may be selected from the family Siphoviridae (such as,
without limitation, genus Camvirus, Likavirus, R4virus, Acadianvirus,
Coopervirus,
Pglvirus, Pipefishvirus, Rosebushvirus, Brujitavirus, Che9cvirus,
Hawkeyevirus,
Plotvirus, Jerseyvirus, Klgvirus, 5p31virus, Lmdlvirus, Una4virus, Bongovirus,
Reyvirus, Buttersvirus, Charlievirus, Redivirus, Baxtervirus, Nymphadoravirus,
Bignuzvirus, Fishburnevirus, Phayoncevirus, Kp36virus, Roguelvirus, Rtpvirus,
Ti virus,
Tlsvirus, Abl8virus, Amigovirus, Anatolevirus, Andromedavirus, Attisvirus,
Barnyardvirus, Bernall3virus, Biseptimavirus, Bronvirus, C2virus, C5virus,
Cbal8lvirus, Cbastvirus, Cecivirus, Che8virus, Chivirus, Cjwlvirus,
Corndogvirus,
Cronusvirus, D3112virus, 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, Nplvirus, Omegavirus, P12002virus, P12024virus, P23virus,
P7Ovirus,
Pa6virus, Pamx74virus, Patiencevirus, Pbilvirus, Pepy6virus, Pfrlvirus,
Phic31virus,
Phicbkvirus, Phietavirus, Phifelvirus, Phijllvirus, Pis4avirus, Psavirus,
Psimunavirus,
Rdjlvirus, Rer2virus, Sap6virus, Send513virus, Septima3virus, Seuratvirus,
Sextaecvirus,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
54
Sfil lvirus, Sfi2ldtivirus, Sitaravirus, Sklvirus, Slashvirus, Smoothievirus,
Soupsvirus,
Spbetavirus, Ssp2virus, T5virus, Tankvirus, Tin2virus, Titanvirus, Tm4virus,
Tp21virus,
Tp84virus, Triavirus, Trigintaduovirus, Vegasvirus, Vendettavirus, Wbetavirus,
Wildcatvirus, Wizardvirus, Woesvirus, XplOvirus, Ydn12virus and Yuavirus).
[138] Bacteriophages may be selected from the family Ackermannviridae (such
as,
without limitation, genus Ag3virus, Limestonevirus, Cba12Ovirus and Vilvirus).
[139] 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,
Inovirus,
Lineavirus, Plectrovirus, Saetivirus, Vespertiliovirus), family Cystoviridae
(such as genus
Cystovirus), family Leviviridae (such as genus Allolevivirus, Levivirus),
family
Microviridae (such as genus Alpha3microvirus, G4microvirus, Phix174microvirus,
Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus) and family
Plasmaviridae
(such as genus Plasmavirus).
[140] 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, Salterprovirus and Bicaudaviridae.
[141] 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
branched 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.
[142] Bacteria of the genus Actinomyces can be infected by the following
phages:
Av-I, Av-2, Av-3, BF307, CT1, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.
[143] Bacteria of the genus Aeromonas can be infected by the following
phages:
AA-I, Aeh2, N, PM1, TP446, 3,4, 11, 13, 29, 31, 32, 37, 43, 43-10T, 51, 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.
[144] Bacteria of the genus Bacillus can be infected by the following
phages: A,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
aizl, Al-K-I, B, BCJA1, BC1, BC2, BLL1, BL1, BP142, BSL1, BSL2, BS1, BS3, BS8,
BS15, BS18, BS22, BS26, BS28, BS31, BS104, BS105, BS106, BTB, B1715V1, C, CK-
I, Coll, Corl, CP-53, CS-I, CSi, D, D, D, D5, entl, FP8, FP9, FSi, F52, F53,
F55, F58,
F59, G, GH8, GT8, GV-I, GV-2, GT-4, g3, g12, g13, g14, g16, g17, g21, g23,
g24, g29, H2,
kenl, KK-88, Kuml, Kyul, J7W-1, LP52, (syn= LP-52), L7, Mexl, MJ-I, mor2, MP-
7,
MP10, MP12, MP14, MP15, Neol, N 2, N5, N6P, PBC1, PBLA, PBP1, P2, S-a, SF2,
SF6,
Shal, Sill, 5P02, (syn= (I)SPP1), 5P13, STI, STi, SU-Il, t, TbI, Tb2, Tb5,
TbIO, Tb26,
Tb51, Tb53, Tb55, Tb77, Tb97, Tb99, Tb560, Tb595, Td8, Td6, Td15, TgI, Tg4,
Tg6,
Tg7, Tg9, TgIO, TgIl, Tg13, Tg15, Tg21, Tinl, Tin7, Tin8, Tin13, Tm3, Tocl,
Togl, toll,
TP-I, TP-10vir, TP-15c, TP-16c, TP-17c, TP-19, TP35, TP51, TP-84, Tt4, Tt6,
type A,
type B, type C, type D, type E, TO, VA-9, W, wx23, wx26, Yunl, a, y, pllõ ymed-
2, (pT,
(pp.-4, (3T, (p75, 005, (syn= 005), 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, AR1,
AR2, AR3,
AR7, AR9, Bace-11, (syn= 11), Bastille, BL1, BL2, BL3, BL4, BLS, BL6, BL8,
BL9,
BP124, B528, B580, Ch, CP-51, CP-54, D-5, darl, denl, DP-7, entl, FoSi, FoS2,
F54,
F56, F57, G, gall, gamma, GE1, GF-2, GSi, GT-I, GT-2, GT-3, GT-4, GT-5, GT-6,
GT-7,
GV-6, g15, 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, PBS1, PK1, PMB1, PMB12, PMJ1, S, SP01, 5P3, 5P5, 5P6,
5P7,
5P8, 5P9, SP10, SP-15, 5P50, (syn= SP-50), 5P82, SST, subl, SW, Tg8, Tg12,
Tg13,
Tg14, thul, thuA, thuS, Tin4, Tin23, TP-13, TP33, TP50, TSP-I, type V, type
VI, V, Vx,
(322, ye, (pNR2, (p25, (p63, 1, 1, 2, 2C, 3NT, 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, B5107, Bl, B2,
GA-I,
GP-I0, GV-3, GV-5, g8, MP20, MP27, MP49, Nf, PPS, PP6, SF5, Tg18, TP-I,
Versailles,
y15, (p29, 1-97, 837/IV, mi-Bacillus (1), Bat10, BSL10, BSLI1, B56, BSI 1,
BS16, B523,
BS101, B5102, g18, morl, PBL1, 5N45, thu2, thu3, TmI, Tm2, TP-20, TP21, TP52,
type F,
type G, type IV, HN-BacMus (3), BLE, (syn= 0c), B52, B54, B55, B57, B10, B12,
B520, B521, 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 1.4..
[145] Bacteria of the genus Bacteriodes can be infected by the following
phages: ad
12, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, Fl, (31, yAl, yBrOl, yBr02,
11, 67.1,
67.3, 68.1, mt-Bacteroides (3), Bf42, Bf71, HN-Bdellovibrio (1) and BF-41.
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
56
[146] Bacteria of the genus Bordetella can be infected by the following
phages: 134
and NN-Bordetella (3).
[147] Bacteria of the genus Borrellia can be infected by the following
phages: NN-
Borrelia (1) and NN-Borrelia (2).
[148] Bacteria of the genus Brucella can be infected by the following
phages: A422,
Bk, (syn= Berkeley), BM29, F0i, (syn= F01), (syn= FQ1), D, FP2, (syn= FP2),
(syn=
FD2), Fz, (syn= Fz75/13), (syn= Firenze 75/13), (syn= Fi), Fi, (syn= F1), Fim,
(syn=
FIm), (syn= Fim), FiU, (syn= FlU), (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),
I, Im, M,
MC/75, M51, (syn= M85), P, (syn= D), S708, 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=
Fi0), (syn= F10), 371/XXIX, (syn= 371), (syn= Fn), (syn= F11) and 513.
[149] Bacteria of the genus Burkholderia can be infected by the following
phages:
CP75, NN-Burkholderia (1) and 42.
[150] 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), VW-3, V2, V3, V8, V16, (syn= Vfi-1),
V19,
V20(V45), V45, (syn= V-45) and NN-Campylobacter (1).
[151] Bacteria of the genus Chlamydia can be infected by the following
phages:
Chpl.
[152] Bacteria of the genus Clostridium can be infected by the following
phages:
CAK1, CAS, Ca7, CEP, (syn= 1C), CEy, Cldl, c-n71, c-203 Tox-, DEP, (syn= ID),
(syn=
1Dt0X+), HM3, KM1, KT, Ms, NA1, (syn= Naltox+), PA1350e, Pf6, PL73, PL78,
PL81,
Pl, P50, P5771, P19402, 1Ct0X+, 2Ct0X\ 2D3 (syn= 2Dt0X+), 3C, (syn= 3Ctox+),
4C,
(syn= 4Ct0X+), 56, III-1, NN-Clostridium (61), NB1t0X+, al, CA1, HMT, HM2,
PF15 P-
23, P-46, Q-05, Q-oe, Q-16, Q-21, Q-26, Q-40, Q-46, S111, SA02, WA01, WA03,
Wm,
W523, 80, C, CA2, CA3, CPT1, CPT4, cl, c4, c5, HM7, H11/A1, H18/Ax, FWS23,
Hi58ZA1, K2ZA1, K21ZS23, ML, NA2t0X; Pf2, Pf3, Pf4, S9ZS3, S41ZA1, S44ZS23,
a2, 41, 112ZS23, 214/S23, 233/Ai, 234/S23, 235/S23, II-1, II-2, II-3, NN-
Clostridium
(12), CA1, Fl, K, S2, 1, 5 and NN-Clostridium (8).
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
57
[153] Bacteria of the genus Corynebacterium can be infected by the
following
phages: CGK1 (defective), A, A2, A3, A101, A128, A133, A137, A139, A155, A182,
B,
BF, B17, B18, B51, B271, B275, B276, B277, B279, B282, C, capi, CC1, CG1, CG2,
CG33, CL31, Cog, (syn= CG5), D, E, F, H, H-I, hqi, hq2, 11ZH33, Ii/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, RS29, S, T, U, UB1, ub2, UH1, UH3, uh3, uh5, uh6, (3, (syn=
(3tox+),
(3hv64, (3vir, y, (syn= ytox-), y19, 6, (syn= 6'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.
[154] Bacteria of the genus Enterococcus can be infected by the following
phages:
DF78, Fl, F2, 1, 2, 4, 14, 41, 867, D1, SB24, 2BV, 182, 225, C2, C2F, E3, E62,
DS96,
H24, M35, P3, P9, SB101, S2, 2B1I, 5, 182a, 705, 873, 881, 940, 1051, 1057,
21096C,
NN-Enterococcus (1), PE1, Fl, F3, F4, VD13, 1, 200, 235 and 341.
[155] Bacteria of the genus Erysipelothrix can be infected by the following
phage:
NN-Eiysipelothrix (1).
[156] Bacteria of the genus Escherichia can be infected by the following
phages:
BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM,
Mu,
(syn= mu), (syn= MuI), (syn= Mu-I), (syn= MU-I), (syn= MuI), (syn= 1,t), 025,
PhI-5, Pk,
PSP3, Pl, P1D, P2, P4 (defective), Sl, Wy, (K13, yR73 (defective), yl, (p2,
(p7, (p92, w
(defective), 7 A, 8y, 9y, 15 (defective), 18, 28-1, 186, 299, HH-Escherichia
(2), AB48,
CM, C4, C16, DD-VI, (syn= Dd-Vi), (syn= DDVI), (syn= DDVi), E4, E7, E28, FIl,
FI3,
H, H1, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, Ox-I (syn= OX1),
(syn=
HF), Ox-2 (syn= 0x2), (syn= 0X2), Ox-3, Ox-4, Ox-5, (syn= 0X5), Ox-6, (syn=
66F),
(syn= y66t), (syn= (66t-)5 0111, PhI-I, RB42, RB43, RB49, RB69, S, Sal-I, Sal-
2, Sal-3,
Sal-4, Sal-5, Sal-6, TC23, TC45, Tull*-6, (syn= TuII*), TuIP-24, TuII*46, TuIP-
60, T2,
(syn= ganuTia), (syn= y), (syn= PC), (syn= P.C.), (syn= T-2), (syn= T2), (syn=
P4), T4,
(syn= T-4), (syn= T4), T6, T35, al, 1, IA, 3, (syn= Ac3), 3A, 3T+, (syn= 3),
(syn= MD,
5y, (syn= (p5), 9266Q, CF0103, HK620, J, K, K1F, m59, no. A, no. E, no. 3, no.
9, N4,
sd, (syn= Sd), (syn= SD), (syn= Sa)3 (syn= sd), (syn= SD), (syn= CD), T3,
(syn= T-3),
(syn= T3), T7, (syn= T-7), (syn= T7), WPK, W31, AH, (C3888, (K3, (K7, (K12, (V-
1,
(1)04-CF, (1)05, (1)06, (1)07, yl, y1.2, (p20, (p95, (p263, y1092, yl, yll,
(syn=cpW), S28, 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, EC1, EC2, E21, E29, Fl, F265, F275, Hi, HK022, HK97, (syn=
(1)HK97), HK139, HK253, HK256, K7, ND-I, no.D, PA-2, q, S2, Tl, (syn= a),
(syn=
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
58
P28), (syn= T-I), (syn= Tx), T3C, T5, (syn= T-5), (syn= T5), UC-I, w, (34, y2,
2\., (syn=
lambda), (syn=(1)k),410326, yy,41006,4107, (1O, ()80, x, (syn= xi), (syn= yx),
(syn= yxi),
2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57,
AC81,
AC95, HK243, K10, ZG/3A, 5, 5A, 21EL, H19-J and 933H.
[157] 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.
[158] Bacteria of the genus Haemophilus can be infected by the following
phages:
HP1, S2 and N3.
[159] Bacteria of the genus Helicobacter can be infected by the following
phages:
HP1 and AA-Helicobacter (1).
[160] Bacteria of the genus Klebsiella can be infected by the following
phages: AI0-
2, KI4B, K16B, K19, (syn= K19), K114, K115, K121, K128, K129, KI32, K133,
K135,
K1106B, K1171B, K1181B, K1832B, A10-I, AO-I, A0-2, A0-3, FC3-10, K, K11, (syn=
KI1), K12, (syn= K12), K13, (syn= K13), (syn= K1 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, K1171B, II, VI, IX, CI-I, K14B, K18,
K111, K112,
K113, K116, K117, K118, K120, K122, K123, K124, K126, K130, K134, K1106B,
KIi65B,
K1328B, KLXI, K328, P5046, 11, 380,111, IV, VII, VIII, FC3-11, K12B, (syn=
K12B),
K125, (syn= K125), K142B, (syn= K142), (syn= K142B), K1181B, (syn= KI181),
(syn=
K1181B), K1765/!, (syn= K1765/1), K1842B, (syn= K1832B), K1937B, (syn=
K1937B),
Ll, (p28, 7, 231, 483, 490, 632 and 864/100.
[161] Bacteria of the genus Lepitospira can be infected by the following
phages:
LE1, LE3, LE4 and -NN-Leptospira (1).
[162] 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, BI 10,
B545, B604, B653, C707, D441, HS047, HlOG, H8/73, H19, H21, H43, H46, H107,
H108, HI10, H163/84, H312, H340, H387, H391/73, H684/74, H924A, PSA, U153,
(pMLUP5, (syn= P35), 00241, 00611, 02971A, 02971C, 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, 11355C, 11711A, 12029, 12981,
13441,
90666, 90816, 93253, 907515, 910716 and NN-Lisferia (15).
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
59
[163] Bacteria of the genus Morganella can be infected by the following
phage: 47.
[164] Bacteria of the genus Mycobacterium can be infected by the following
phages:
13, AG1, ALi, ATCC 11759, A2, B.C3, BG2, BK1, BK5, butyricum, B-I, B5, B7,
B30,
B35, Clark, Cl, C2, DNAIII, DSP1, D4, D29, GS4E, (syn= GS4E), GS7, (syn= GS-
7),
(syn= G57), IPa, lacticola, Legendre, Leo, L5, (syn= TL-5), MC-I, MC-3, MC-4,
minetti,
MTPHIl, Mx4, MyF3P/59a, phlei, (syn= phlei 1), phlei 4, Polonus II,
rabinovitschi,
smegmatis, TM4, TM9, TM10, TM20, Y7, Y10, (p630, IB, IF, IH, 1/1, 67, 106,
1430, Bl,
(syn= Bol), B24, D, D29, F-K, F-S, HP, Polonus I, Roy, R1, (syn= Rl-Myb),
(syn= Ri),
11, 31, 40, 50, 103a, 103b, 128, 3111-D, 3215-D and NN-Mycobacterium (1).
[165] Bacteria of the genus Neisseria can be infected by the following
phages:
Group I, group II and NP1.
[166] Bacteria of the genus Nocardia can be infected by the following
phages:
MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.
[167] 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,
PmI, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, PmIl, Pv2, al, pm, 7/549, 9B/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, n2600, yx7, 1/1004, 5/742, 9,
12, 14, 22,
24/860, 2600/D52, Pm8 and 24/2514.
[168] Bacteria of the genus Providencia can be infected by the following
phages:
PL25, PL26, PL37, 9211/9295, 9213/921 Ib, 9248, 7/R49, 7476/322, 7478/325,
7479,
7480, 9000/9402 and 9213/921 Ia.
[169] Bacteria of the genus Pseudomonas can be infected by the following
phages:
PH, (syn= Pf-I), Pf2, Pf3, PP7, PRR1, 7s, im-Pseudomonas (1), AI-I, AI-2, B
17, B89,
CB3, Col 2, Col 11, Col 18, Col 21, C154, C163, C167, C2121, E79, F8, ga, gb,
H22, Kl,
M4, N2, Nu, PB-I, (syn= PB1), pf16, PMN17, PP1, PP8, Psal, PsPl, PsP2, PsP3,
PsP4,
PsP5, P53, PS17, PTB80, PX4, PX7, PY01, 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, P1K, SLP1, 5L2, S2, UNL-I, wy, Yai, Ya4, Yan, (BE, (pCTX, (C17,
yKZ, (syn=41)1(Z), (p-LT,410mu78, (NZ, (pPLS-1, yST-1, (W-14, (p-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, 12S,
16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188, 337, 352, 1214, HN-
Pseudomonas (23),
A856, B26, CI-I, CI-2, C5, D, gh-1, Fl 16, HF, H90, K5, K6, K1 04, K109, K166,
K267,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
N4, N5, 06N-25P, PE69, Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6,
PP7,
PP8, PP56, PP87, PP1 14, PP206, PP207, PP306, PP651, Psp231a, Pssy401,
Pssy9220,
psi, PTB2, PTB20, PTB42, PX1, PX3, PX10, PX12, PX14, PY070, PY071, R, SH6,
SH133, tf, Ya5, Ya7, (BS,411(f77, (p-MC, (tomnF82, (pPLS27, (pPLS743, (S-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-I, C22, D3, D37, D40, D62, D3112, F7, F10, g, gd, ge, g
Hw12, Jb
19, KF1, L , OXN-32P, 06N-52P, PCH-I, PC13-1, PC35-1, PH2, PH51, PH93, PH132,
PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PM1 13, PM681, PM682,
PO4, PP1, PP4, PP5, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711,
PP891,
Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-I0, Pz, SD1, SL1,
SL3,
SL5, SM, (C5, (C11, (C11-1, (C13, (C15, (M0, (pX, (p04, q11, (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, Pssy15, Pssy4210, Pssy4220, PY012, PY034, PY049, PY050,
PY051, PY052, PY053, PY057, PY059, PY0200, PX2, PX5, 5L4, (p03, (p06 and
1214.
[170] Bacteria of the genus Rickettsia can be infected by the following
phage: NN-
Rickettsia.
[171] 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= 04), (syn= 7), 02, 03, P3, P9a, P10, 5ab3, 5ab5, San1S,
5an17, SI,
Taunton, Vii, (syn= Vii), 9, imSalmonella (1), N-I, N-5, N-I0, 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, ViIV, ViV, ViVI, ViVII, Worksop, 5j5, 634,
1,37,
1(40), (syn= (p1[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, G1
73, h, IRA, Jersey, MB78, P22-1, P22-3, P22-12, Sabi, 5ab2, 5ab2, 5ab4, Sanl,
5an2,
5an3, 5an4, 5an6, 5an7, 5an8, 5an9, 5an13, 5an14, 5an16, 5an18, 5an19, 5an20,
5an21,
5an22, 5an23, 5an24, 5an25, 5an26, SasL1, SasL2, SasL3, SasL4, SasL5, S1BL,
SIT, Viii,
(pl, 1, 2, 3a, 3al, 1010, Ym-Salmonella (1), N-4, SasL6 and 27.
[172] Bacteria of the genus Serratia can be infected by the following
phages: A2P,
PS20, SMB3, SMP, SMP5, 5M2, V40, V56, ic, (I)CP-3, (I)CP-6, 3M, 10/1a, 20A,
34CC,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
61
34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232,
L2232, L34, L.228, SLP, SMPA, V.43, a, (pCW1,41)CP6-1,41)CP6-2,41)CP6-5, 3T,
5, 8,
9F, 10/1, 20E, 32/6, 34B, 34CT, 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 SMB1.
[173] 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, 17.8, H-Sh,
PE5,
P90, Sf1I, Sh, SHm, SHrv, (syn= HIV), SHvi, (syn= HVI), SHVvm, (syn= HVIII),
SK766, (syn= gamma 66), (syn= yf3f3), (syn= y66b), SKm, (syn= SIIIb)5 (syn=
UI), SKw,
(syn= Siva), (syn= IV), SIC TM , (syn= SIVA.), (syn= IVA), SKvi, (syn= KVI),
(syn= Svi),
(syn= VI), SKvm, (syn= Svm), (syn= VIII), SKVIIIA, (syn= SvmA), (syn= VIIIA),
STvi,
STK, STxl, STxn, S66, W2, (syn= D2c), (syn= D20), yl, ylVb 3-SO-R, 8368-SO-R,
F7,
(syn= F57), (syn= K29), F10, (syn= FS10), (syn= K31), Ii, (syn= alfa), (syn=
FSa),
(syn= K1 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), SHv, (syn=
SHV),
SHx, SHX, SKn, (syn= K2), (syn= KII), (syn= Sn), (syn= SsII), (syn= II), SKrv,
(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= T35), (syn= 35-50-R), STvm, (syn=
T8345), (syn= 8345-SO-S-R), Wl, (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= Ssy66), (p2, BIl, 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), SHva, (syn= HVII),
SHK, (syn= HIX), SHxl, SHx7c, (syn= HXn), SKI, KI, (syn= Si), (syn= SsI),
SKVII,
(syn= KVII), (syn= Sva), (syn= SsVII), SKIX, (syn= KIX), (syn= Six), (syn=
SsIX),
SKXII, (syn= KXII), (syn= Sxn), (syn= SsXII), STi, STffl, STrv, STVi, STva,
S70, S206,
U2-S0-S, 3210-SO-S, 3859-SO-S, 4020-SO-S, (p3, (p5, (p7, (p8, (p9, y10, q1 1,
y13, y14,
y18, SHm, (syn= Hai), SHri, (syn= HXt) and SKxI, (syn= KXI), (syn= Sri), (syn=
SsXI),
(syn= XI).
[174] Bacteria of the genus Staphylococcus can be infected by the following
phages:
A, EW, K, Ph5, Ph9, PhIO, Ph13, Pl, P2, P3, P4, P8, P9, P10, RG, SB-i, (syn=
Sb-I), S3K,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
62
Twort, (I)SK311, (p812, 06, 40, 58, 119, 130, 131, 200, 1623, STC1,
(5yn=5tc1), STC2,
(syn=stc2), 44AHJD, 68, Ad, AC2, A6"C", A9"C", b581, CA-I, CA-2, CA-3, CA-4,
CA-5, DI1, L39x35, L54a, M42, Ni, N2, N3, N4, N5, N7, N8, NiO, Nil, N12, N13,
N14,
N16, Ph6, Ph12, Ph14, UC-18, U4, U15, Si, S2, S3, S4, S5, X2, Z1, (B5-2, (pD,
0), 11,
(syn= yl 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= 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,
2638C,
2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100, AC3, A8, A10, A13,
b594n, D, HK2, N9, N15, P52, P87, Si, S6, Z4, (RE, 3A, 3B, 3C, 6, 7, 16, 21,
42B, 42C,
42E, 44, 47, 47A5 47C, 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).
[175] Bacteria of the genus Streptococcus can be infected by the following
phages:
EJ-I, NN-Streptococais (1), a, Cl, FLOThs, H39, Cp-I, Cp-5, Cp-7, Cp-9, Cp-I0,
AT298,
AS, alO/J1, alO/J2, alO/J5, alO/J9, A25, BTI1, b6, CA1, c20-1, c20-2, DP-I, Dp-
4, DT1,
ET42, el0, FA101, FEThs, Fic, FKKIOI, FKLIO, FKP74, FKH, FLOThs, FyI01, fl,
F10,
F20140/76, g, GT-234, HB3, (syn= HB-3), HB-623, HB-746, M102, 01205, y01205,
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, Sfil 1, (syn= SFiI1), (syn= (pSFill), (syn= (Will), (syn= (pSfil 1),
sfil9, (syn= SFil9),
(syn= (pSFil9), (syn= (p5fil9), 5fi21, (syn= SFi21), (syn= (pSFi21), (syn=
(p5fi21), STO,
STX, st2, 5T2, 5T4, S3, (syn= 03), s265, (1)17, (p42, (1)57, y80, (p81, (p82,
(p83, (p84, (p85,
(p86, (p87, (p88, (p89, y90, (p91, (p92, (p93, (p94, (p95, (p96, (p97, (p98,
(p99, y100, y101,
y102, (p227, (1)7201, wl, w2, w3, w4, w5, w6, w8, w10, 1, 6, 9, 10F, 12/12,
14, 175R, 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, 80/5T16, 80/15, 80/47, 80/48, 101, 103/39, 103/40, 121/41,
121/42, 123/43,
123/44, 124/44, 337/5T17 and mStreptococcus (34).
[176] Bacteria of the genus Treponema can be infected by the following
phage: NN-
Treponema (1).
[177] Bacteria of the genus Vibrio can be infected by the following phages:
CTX41),
fs, (syn= si), fs2, Ivpf5, Vf12, Vf33, VPI41), VSK, v6, 493, CP-T1, ET25,
kappa, K139,
Labol, )XN-69P, OXN-86, 06N-21P, PB-I, P147, rp-1, 5E3, VA-I, (syn= VcA-I),
VcA-
2, VP1, VP2, VP4, VP7, VP8, VP9, VP10, VP17, VP18, VP19, X29, (syn= 29
d'Herelle),
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
63
t, (DHAWI-1, (DHAWI-2, (DHAWI-3, (DHAWI-4, (DHAWI-5, (DHAWI-6, (DHAWI-7,
XHAWI-8, (1)HAWI-9, (1)HAWI-10, (1)HC1-1, (1)HC1-2, (1)HC1-3, (1)HC1-4, (1)HC2-
1,
>HC2-2, (1)HC2-3, (1)HC2-4, (1)HC3-1, (1)HC3-2, (1)HC3-3, (1)HD1S-1, (1)HD1S-
2,
(DHD2S-1, (DHD2S-2, (DHD2S-3, (DHD2S-4, (DHD2S-5, (DHDO-1, (DHDO-2, (DHDO-3,
(DHDO-4, (DHDO-5, (DHDO-6, (DKL-33, (DKL-34, (DKL-35, (DKL-36, (DKWH-2,
(DKWH-3, (DKWH-4, (DMARQ-1, (DMARQ-2, (DMARQ-3, (DMOAT-1, (D0139,
(1)PEL1A-1, (1)PEL1A-2, (1)PEL8A-1, (1)PEL8A-2, (1)PEL8A-3, (1)PEL8C-1,
(1)PEL8C-2,
(1)PEL13A-1, (1)PEL13B-1, (1)PEL13B-2, (1)PEL13B-3, (1)PEL13B-4, (1)PEL13B-5,
(1)PEL13B-6, (1)PEL13B-7, (1)PEL13B-8, (1)PEL13B-9, (1)PEL13B-10, yVP143,
yVP253,
(D16, (p138, 1- II, 5, 13, 14, 16, 24, 32, 493, 6214, 7050, 7227, II, (syn=
group II), (syn==
(p2), V, VIII, -m-Vibrio (13), KVP20, KVP40, nt-1, 06N-22P, P68, el, e2, e3,
e4, e5, FK,
G, I, K, nt-6, Ni, N2, N3, N4, N5, 06N-34P, OXN-72P, OXN-85P, OXN-100P, P, Ph-
I,
PL163/10, Q, S, T, (p92, 1-9, 37, 51, 57, 70A-8, 72A-4, 72A-10, 110A-4, 333,
4996,1
(syn= group I), III (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-8,110A-1, 110A-
5,
110A-7, hv-1, OXN-52P, P13, P38, P53, P65, P108, Pill, TP13 VP3, VP6, VP12,
VP13,
70A-3, 70A-4, 70A-10, 72A-1, 108A-3, 109-B1, 110A-2, 149, (syn= y149), IV,
(syn=
group IV), NN-Vibrio (22), VP5, VPI1, VP15, VP16, al, a2, a3a, a3b, 353B and
HN-
Vibrio (7).
[178] 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/C753-76, 8/C239-76, 9/F18167, 1701, 1710, PST, 1/F2852-
76,
D'Herelle, EV, H, Kotljarova, PTB, R, Y, YerA41, yYer03-12, 3, 4/C1324-76,
7/F783-
76, 903, 1/M6176 and Yer2AT.
[179] In an embodiment, the bacteriophage is selected in the group
consisting of
Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya
virus
RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus
38,
Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10,
Salmonella virus
SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4,
Salmonella virus Marshall, Salmonella virus Maynard, Salmonella virus SJ2,
Salmonella
virus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiella virus
0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus
CP21,
Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus
IBB35,
Campylobacter virus CP81, Campylobacter virus CP30A, Campylobacter virus CPX,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
64
Campylobacter virus NCTC12673, Erwinia virus Ea214, Erwinia virus M7,
Escherichia
virus AY0145A, Escherichia virus EC6, Escherichia virus HY02, Escherichia
virus JH2,
Escherichia virus TP1, Escherichia virus VpaEl, 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
L413C, Staphylococcus virus Gl, Staphylococcus virus G15, Staphylococcus virus
JD7,
Staphylococcus virus K, Staphylococcus virus MCE2014, 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 SAll, Staphylococcus virus 5tau2, Bacillus
virus
Camphawk, Bacillus virus SP01, Bacillus virus BCP78, Bacillus virus TsarBomba,
Staphylococcus virus Twort, Enterococcus virus phiEC24C, Lactobacillus virus
Lb338-1,
Lactobacillus virus LP65, Enterobacter virus PG7, Escherichia virus CC31,
Klebsiella
virus JD18, Klebsiella virus PK0111, Escherichia virus Bp7, Escherichia virus
IME08,
Escherichia virus JS10, Escherichia virus J598, 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 phil, 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 ntl, Vibrio virus ValKK3, Escherichia virus VR7, Escherichia virus VR20,
Escherichia virus VR25, Escherichia virus VR26, Shigella virus 5P18,
Escherichia virus
AR1, Escherichia virus C40, Escherichia virus E112, Escherichia virus ECML134,
Escherichia virus HY01, Escherichia virus Ime09, Escherichia virus RB3,
Escherichia
virus RB14, Escherichia virus T4, Shigella virus Pssl, Shigella virus 5hfl2,
Yersinia virus
D1, Yersinia virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas
virus
Aehl, Escherichia virus RB16, Escherichia virus RB32, Escherichia virus RB43,
Pseudomonas virus 42, Cronobacter virus CR3, Cronobacter virus CR8,
Cronobacter
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
virus CR9, Cronobacter virus PBES02, Pectobacterium virus phiTE, Cronobacter
virus
GAP31, Escherichia virus 4MG, Salmonella virus SE1, Salmonella virus 55E121,
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 5immy50, 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 Bcpl, Bacillus virus BCP82, Bacillus virus BM15,
Bacillus virus
Deepblue, Bacillus virus JBP901, Burkholderia virus Bcepl, 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 Bxzl, 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 Grass, Bacillus
virus
NIT1, Bacillus virus 5PG24, Aeromonas virus 43, Escherichia virus Pl,
Pseudomonas
virus CAbl, Pseudomonas virus CAb02, Pseudomonas virus JG004, Pseudomonas
virus
PAKP1, Pseudomonas virus PAKP4, Pseudomonas virus PaP1, Burkholderia virus
BcepFl, 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
66
Ralstonia virus RSL1, Aeromonas virus 25, Aeromonas virus 31, Aeromonas virus
Aes12, Aeromonas virus Aes508, Aeromonas virus AS4, 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 Abpl, Acinetobacter virus Fril, Acinetobacter virus
IME200,
Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobacter virus
phiAB1,
Escherichia virus K30, Klebsiella virus K5, Klebsiella virus K11, Klebsiella
virus Kpl,
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
5U552A, 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 KlE, Salmonella virus 5P6, Escherichia virus T7, Kluyvera
virus Kvpl,
Pseudomonas virus ghl, Prochlorococcus virus PSSP7, Synechococcus virus P60,
Synechococcus virus Syn5, Streptococcus virus Cpl, Streptococcus virus Cp7,
Staphylococcus virus 44AHJD, Streptococcus virus Cl, Bacillus virus B103,
Bacillus
virus GA1, Bacillus virus phi29, Kurthia virus 6, Actinomyces virus Avl,
Mycoplasma
virus Pl, Escherichia virus 24B, Escherichia virus 933W, Escherichia virus
Min27,
Escherichia virus PA28, Escherichia virus 5tx2 II, Shigella virus 75025tx,
Shigella virus
POCJ13, Escherichia virus 191, Escherichia virus PA2, Escherichia virus
TL2011,
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 Sbl,
Achromobacter
virus Axp3, Achromobacter virus JWAlpha, Edwardsiella virus KF1, Pseudomonas
virus
KPP25, Pseudomonas virus R18, Pseudomonas virus Ab09, Pseudomonas virus LIT1,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
67
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 5T64T, 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 Septimall, Escherichia virus SU10, Brucella virus Pr, Brucella 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 SIOl, 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 Izzy, 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 Banel, 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 Pgl, 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
Entl,
Salmonella virus f18SE, 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
68
virus wks13, Escherichia virus K1G, Escherichia virus K1H, Escherichia virus
Klindl,
Escherichia virus Klind2, Salmonella virus 5P31, Leuconostoc virus Lmdl,
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, 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 Roguel, Escherichia virus ACGM12, Escherichia virus
Rtp,
Escherichia virus ADB2, Escherichia virus JMPW1, Escherichia virus JMPW2,
Escherichia virus Ti, Shigella virus PSf2, Shigella virus Shfll, 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 Faithl, 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
69
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 SG4,
Mycobacterium virus Shaunal, 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, 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 S01, 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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
Billknuckles,
Mycobacterium virus Bruns, Mycobacterium virus Bxbl, 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 JC27, 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, 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 JenKl,
Escherichia virus
JenPl, 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
71
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 PlOOD, 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, Propionibacterium virus Pirate,
Propionibacterium
virus Procrassl, Propionibacterium virus SKKY, Propionibacterium virus Solid,
Propionibacterium virus Stormborn, Propionibacterium virus Wizzo, Pseudomonas
virus
PaMx28, Pseudomonas virus PaMx74, Mycobacterium virus Patience, Mycobacterium
virus PBIl, 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
72
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 Send513, 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
Sfill,
Streptococcus virus 7201, Streptococcus virus DT1, Streptococcus virus
phiAbc2,
Streptococcus virus Sfi19, Streptococcus virus Sfi21, Paenibacillus virus
Diva,
Paenibacillus virus Hb10c2, Paenibacillus virus Rani, Paenibacillus virus
Shelly,
Paenibacillus virus Sitara, Paenibacillus virus Willow, Lactococcus virus 712,
Lactococcus virus ASCC191, Lactococcus virus A5CC273, Lactococcus virus
ASCC281,
Lactococcus virus A5CC465, Lactococcus virus A5CC532, Lactococcus virus
Bibb29,
Lactococcus virus bIL170, Lactococcus virus CB13, Lactococcus virus CB14,
Lactococcus virus CB19, Lactococcus virus CB20, Lactococcus virus jj50,
Lactococcus
virus P2, Lactococcus virus P008, Lactococcus virus ski, Lactococcus virus
S14, 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 MARIO, Vibrio virus
55P002,
Escherichia virus AKFV33, Escherichia virus BF23, Escherichia virus DT57C,
Escherichia virus EPS7, Escherichia virus FFH1, Escherichia virus H8,
Escherichia virus
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
73
s1ur09, Escherichia virus T5, Salmonella virus 118970sa12, Salmonella virus
Shivani,
Salmonella virus 5PC35, Salmonella virus Stitch, Arthrobacter virus Tank,
Tsukamurella
virus TIN2, Tsukamurella virus TIN3, Tsukamurella virus TIN4, 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 phil2, Staphylococcus virus phiSLT, Mycobacterium virus
32HC,
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 XplO, Streptomyces virus TP1604,
Streptomyces virus YDN12, Alphaproteobacteria virus phiJ1001, 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 phil2, Pseudomonas virus phil3,
Pseudomonas virus phi2954, Pseudomonas virus phiNN, Pseudomonas virus phiYY,
Vibrio virus fsl, 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 If 1,
Propionibacterium virus B5, Pseudomonas virus Pfl, 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 Cflc,
Spiroplasma
virus C74, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x, Escherichia
virus
Fl, Escherichia virus Qbeta, Escherichia virus BZ13, Escherichia virus M52,
Escherichia
virus a1pha3, Escherichia virus ID21, Escherichia virus ID32, Escherichia
virus ID62,
Escherichia virus NC28, Escherichia virus NC29, Escherichia virus NC35,
Escherichia
virus phiK, Escherichia virus Stl, Escherichia virus WA45, Escherichia virus
G4,
Escherichia virus ID52, Escherichia virus Talmos, Escherichia virus phiX174,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
74
Bdellovibrio virus MAC1, Bdellovibrio virus MH2K, Chlamydia virus Chpl,
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 Wipl,
Escherichia virus phi80, Escherichia virus RB42, Escherichia virus T2,
Escherichia virus
T3, Escherichia virus T6, Escherichia virus VT2-Sa, Escherichia virus VT1-
Sakai,
Escherichia virus VT2-Sakai, Escherichia virus CP-933V, Escherichia virus P27,
Escherichia virus Stx2phi-I, Escherichia virus Stxlphi, Escherichia virus
Stx2phi-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 ECO26 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.
[180] In one embodiment, the bacterial virus particles typically target E.
coli and
include the capsid of a bacteriophage selected in the group consisting of
BW73, B278,
D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, 025, PhI-
5, Pk,
PSP3, Pl, P1D, P2, P4, Sl, Wy, yK13, yl, y2, y7, y92, 7 A, 8y, 9y, 18, 28-1,
186, 299,
HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, E4, E7, E28, FIl, FI3, H, H1,
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,
PhI-I, RB42, RB43, RB49, RB69, S, Sal-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6,
TC23, TC45,
TuII*-6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, al, 1, IA, 3, 3A, 3T+,
5y, 9266Q,
CF0103, HK620, J, K, K1F, m59, no. A, no. E, no. 3, no. 9, N4, sd, T3, T7,
WPK, W31,
AH, yC3888, 2K3, 2K7, 2K12, yV-1,41)04-CF,41005,41006,41007, yl, y1.2, y20,
y95, y263,
y1092, yl, yll, S28, 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, EC1, EC2, E21, E29, Fl,
F26S,
F27S, Hi, HK022, HK97, HK139, HK253, HK256, K7, ND-I, PA-2, q, S2, Tl, ), T3C,
T5, UC-I, w, (34, y2, X, , (I)D326, yy,41006,4107, (MO, y80, x, 2, 4, 4A, 6,
8A, 102, 150, 168,
174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, K10, ZG/3A,
5, 5A, 21EL, H19-J and 933H.
[181] The present disclosure provides pharmaceutical or veterinary
compositions
comprising one or more of the bacterial delivery vehicles disclosed herein and
a
pharmaceutically acceptable carrier. Generally, for pharmaceutical use, the
bacterial
delivery vehicles may be formulated as a pharmaceutical preparation or
compositions
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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. 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
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.
[182] 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 the bacterial delivery vehicles or
compositions
disclosed herein to a subject having a bacterial infection in need of
treatment. In some
embodiments, the subject is a mammal. In some embodiments, the subject is a
human.
[183] 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.
[184] 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 of the disclosure 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
76
polysorbate 8o (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.
[185] The bacterial delivery vehicles according to the disclosure 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 solubilizers,
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
for enteral administration. The liquid vehicle for pressurized compositions
can be a
halogenated hydrocarbon or other pharmaceutically acceptable propellant.
[186] 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.
[187] 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.
[188] 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, conjunctivitis, cholecystitis, colorectal cancer,
polyposis,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
77
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 Widermere
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.
[189] 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, post-partum 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. In another embodiment, the infection according to the
disclosure is
caused by a bacterium expressing toxin, such as shiga-toxin. In a particular
embodiment,
the infection is caused by a Shiga-Toxin producing E. coli (STEC).
[190] 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.
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
78
[191] 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 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-Li (programmed death ligand 1) inhibitor and CTLA-4
(cytotoxic T lymphocyte associated protein 4).
[192] 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.
[193] 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.
[194] 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
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
79
deliver the packaged plasmid. The disclosure also relates to a pharmaceutical
or
veterinary composition according to the disclosure for use in the treatment of
a disease or
disorder such as bacterial infection wherein the pharmaceutical or veterinary
composition
is obtained by the method comprising : i) obtaining a biological sample from
the
individual to be treated 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) preparing the
pharmaceutical
or veterinary composition capable of recognizing each pathogenic bacterial
strain or
species identified in the sample and to deliver the packaged plasmid.
[195] 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.
[196] 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.
[197] In another particular embodiment, the disclosure concerns 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.
[198] 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,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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.
[199] 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 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.
[200] 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, cefaloridine,
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,
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
81
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,
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.
[201] In another particular embodiment, the disclosure concerns the in-situ
bacterial
production of any compound of interest, including therapeutic compound such as
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
82
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.
[202] 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.
[203] The present invention will be further illustrated by the examples
below.
EXAMPLES
EXAMPLE 1
[204] Lambda-packaged cosmids are derived from lambda PaPa, a variant of
the
wild-type Ur-lambda phage with a frameshift mutation in the stf gene [7]
leading to a
truncated protein which is an inactive STF protein, i.e a protein with no
biological
activity. Lambda Papa has been used as the de facto wild-type lambda phage in
the
majority of laboratory studies, because as opposed to wild-type Ur-lambda, it
makes
larger plaques that are easier to handle. The stf gene codes for the side tail
fiber protein,
which in the case of phage lambda recognizes the secondary receptor on the
cell surface,
OmpC.
[205] This secondary receptor allows for transient binding of the phage
particle on
the cell surface in order to scan the surface and position the injection
machinery in
contact with the primary receptor (LamB in the case of lambda, interaction
mediated by
the lambda protein gpJ). Since the STF binding is reversible, it allows the
phage to
"walk" on the cell surface until a primary receptor is found and the infection
process
starts. These protein complexes are sometimes referred to as "L-shape fibers",
such as in
T5, "side tail fibers" such as in lambda, "long tail fibers" as in T4, or
tailspikes such as in
phage P22 [7]¨[10]. For some phages, the presence of this second set of
proteins is
necessary for the infection process to occur, such as T4 [8]. In some other
phages, like
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
83
lambda, this second set of proteins is not necessary for the infection process
to happen,
but it may allow for a more efficient attachment to the target cell [7].
[206] The wild-type length of the lambda phage genome is 48.5 kbp. It is
well
known that lambda can only package DNA from 37.7 kbp to 51 kbp [11], or about
78% to
105% of the wild-type length. Smaller DNA payloads do not build up enough
pressure
inside the capsid for packaging termination to occur and larger ones make the
capsid too
unstable. Additionally, smaller genomes have been shown to be ejected with
much lower
efficiency in the presence of higher external osmotic pressures [12]: as the
length of the
encapsidated DNA decreases, the ejection force decreases in an exponential
fashion
between the two size extremes 37.7 to 51 kbp. It is also known that the
smaller the
payload is, the lower the efficiency at which packaged particles will form
[11].
Combining these two observations, it can be concluded that a smaller DNA
payload will
be detrimental if high enough titers for in vivo experiments are needed.
[207] Most of the phagemids packaged using the lambda system (and many
others)
are much smaller than the wild-type length of the phage. Packaging is possible
because
the cosmid forms concatemers via the sigma replication pathway: when the
concatemers
fall between 74% and 105% of the wild-type lambda genome length, packaging is
terminated and a mature packaged cosmid is formed [11]. This means that these
particles,
in contrast to a wild-type lambda genome, will not have packaged DNA of a
homogeneous length: the whole range of 74% to 105% genome lengths will be
present.
For example, in [11] a cosmid of 12.8 kb was shown to be packaged as trimers
and
tetramers, which correspond to 38.4 kb and 51.2 kb (in the range of allowed
packaged
sizes); the 38.4 kb variant was found in about 15% of the particles while the
51.2 kb was
found in about 40%. Similarly, a 4.6 kb plasmid was shown to be packaged as a
9-mer,
10-mer and 11-mer (41.4 kb, 46 kb and 50.6 kb). In this case, the 41 kb
variant was the
most common with about 20% of the particles having this size, followed by the
50.6 kb
variant with about 12%; the 46 kb variant was only present in about 5% of the
particles.
[208] For in vivo applications, such as oral delivery of encapsidated DNA
particles,
packaged phagemids will need to be given at high enough concentrations to
reach all the
target cells; hence, a payload that gives high enough titers is essential to
optimize the in
vivo activity as well as the manufacturing process.
[209] Finally, besides a payload with a proper size, the packaged particle
needs to be
able to bind its target cell strongly and long enough for the injection
process to occur. It
was previously shown that the presence of STF is not necessary for lambda-
mediated in
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
84
vitro transduction experiments in K-12 laboratory strains [7].
[210] Here, the inventors have unexpectedly shown that both a suitable
payload that
is packaged as concatemers of the correct length as well as a functional side
tail fiber
greatly increase efficiency when performing in vivo delivery assays of lambda-
based
packaged phagemids. The inventors have also unexpectedly demonstrated herein
that for
strains other than model K12 strains, STF and/or gpJ from other phages that
can
specifically recognize surface antigens/receptors of the target bacteria can
be used to
generate engineered lambda viral particles that mediate efficient delivery
both in vitro and
in vivo.
[211] A mouse model, using E. coli strain MG1655, or derivatives, with
engineered
streptomycin resistance was developed using an established colonization
protocol [13].
Mice (Balb/c ByJ, 7 weeks) were treated with a short course of streptomycin,
which is
known to reduce the natural coliform intestinal population [14]. This
treatment allows for
exogenous E. coli to be administered and colonize empty ecological niches
while
maintaining other species that are present in the natural microbiota. In
detail, animals
were treated with a 5-day course of 5g/L streptomycin in drinking water. The
treatment
was stopped 5 days before gavaging of the packaged phagemids to avoid any bias
and
selection of resistance due to antibiotic evolutionary pressure.
[212] To test the in vivo delivery efficiency of packaged phagemids,
psgRNAcos,
the 2.5 kb cosmid of SEQ ID NO: 1 carrying a kanamycin resistance gene was
packaged
into lambda PaPa particles, encoding a non-functional stf gene. To achieve
this, the
cosmid was transformed in an E. coli strain carrying the lambda prophage
lacking a cos
site but otherwise possessing all the machinery for the induction of the
lambda phage lytic
cycle as well as the DNA packaging system. The cI repressor of the lambda
prophage
carries mutations making it thermosensitive and enabling the induction of the
lytic cycle
through temperature change. The cells containing the lambda cosmid were grown
at 30 C
in liquid LB media. At an 0D600 of 0.6, the culture was shifted to 42 C for 25
minutes to
induce the entry into lytic cycle. After that, cells were shifted back to 37 C
for 3 hours to
allow for virion assembly containing the lambda cosmid. Cells were then
centrifuged and
washed in lambda buffer (10 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgSO4).
Chloroform was added and the sample was spun down at 17,000 g for 5 minutes.
Finally,
the aqueous phase was collected and filtered through a 0.2 p.m pore-size
filter. The titer of
the packaged phagemids was measured by performing a transduction assay in
vitro using
strain MG-GFP as a recipient. MG-GFP is a derivative of strain MG1655 with a
gfp
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
fluorescent reporter gene and an ampicillin resistance gene inserted in the
chromosome.
The titer was determined to be about 106 particles/pi (not shown). This
packaged
phagemid stock was used to transduce strain MG-GFP in the gut of mice
colonized by
MG-GFP for 5 days. Feces were collected at different time points, homogenized
and
plated on LB agar plates with kanamycin to monitor the number of transduced MG-
GFP
cells. Kanamycin-resistant colonies were counted in different parts of the
mice intestinal
tract (psgRNAcos encodes a kanamycin marker). As depicted in FIG. 1. almost no
detectable transductants were observed anywhere.
[213] The low delivery efficiencies observed are consistent with the fact
that the
lambda PaPa phage replicates very poorly in the gut of mice colonized by MG-
GFP, as
measured by counting plaque forming units over time in the feces of mice
colonized by
MG-GFP and infected with lambda PaPa. To identify the reason for this poor
efficiency
of lambda PaPa in the mouse gut, an in vivo evolution assay was performed to
select
improved variants of this phage. Mice colonized by MG-GFP were gavaged with an
initial total dose of 1071ambda PaPa. Every day, faeces were collected and
tested for their
ability to form plaques on lawns of MG1655. They were also homogenized in 1X
PBS at
40 mg/mL, filtered through a 0.22 1.tm pore membrane and re-fed to the mice.
As can be
seen in FIG.2, initially there were virtually no PFUs detected, as lambda Papa
was not
able to bind its host in the mouse gut. Some peaks were observed in the amount
of PFU-
forming particles whose amount oscillated from high to undetectable levels up
to day 17,
where a steady increase in the number of PFU was detected.
[214] Strikingly, when the genomes of the phage particles obtained on day
35 were
sequenced, it was observed that the ORF of the stf gene had been restored: the
lambda
PaPa particles had reverted the mutation to give a full-length stf gene
(although with
mutations in K338E, T391M and A3955 with respect to the canonical STF sequence
of
SEQ ID NO: 14). Additionally, it was observed that the gpJ gene, responsible
for binding
to the LamB receptor, had also mutated in three positions. These mutations
were tested to
determine if they altered the receptor being used by this phage on wild-type
MG1655 and
MG1655-delta-LamB. The inventors saw no difference, which confirmed that the
new
Ur-lambda particles still use LamB as their primary receptor.
[215] With the ORF of the stf gene restored in the original lambda packaged
phagemid production strain, but keeping the original STF sequence of SEQ ID
NO: 14
and not the mutated version obtained in the experiment above, the same
experiment was
performed with a packaged phagemid containing a DNA payload of 3 kb in size
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
86
(pJ23104-GFP of SEQ ID NO: 2) but this time encoding a chloramphenicol marker.
The
sequence of the gpJ gene was not changed at this stage. The packaged phagemids
were
gavaged to mice colonized with MG1655-Strp (with streptomycin resistance).
Feces were
homogenized at 40 mg/mL in 1X PBS and serially diluted and plated in Drigalski
plates
supplemented or not with 25 1.tg/mL chloramphenicol. The amount of
chloramphenicol
resistant cells was hence counted in the faeces up to 48 h after transduction.
As can be
seen in FIG.3, the amount of transduced cells increased as compared to lambda
PaPa
packaged phagemids, but the delivery efficiency was very low (about 1 in 1000
cells).
When the same experiment was attempted with packaged Ur-lambda particles with
a
modified gpJ gene to mimic the particles obtained in the in vivo evolution
assay, the same
results were observed. It was concluded that there must be another factor that
prevents the
packaged phagemids from working optimally in vivo, as no significant
differences were
seen in in vitro transduction assays.
[216] An experiment was then done in which a packaged phagemid was produced
with a larger DNA payload, pJF1 of SEQ ID NO: 3 (around 7 kb) in Ur-lambda
capsids
and administered to the mice. It is important to note that the gpJ gene was
not changed, so
these particles encode the wild-type gpJ lambda of SEQ ID NO: 10 and still use
LamB as
their primary receptor. The same protocol was followed for in vivo delivery as
for the
smaller DNA payload of SEQ ID NO: 2. Surprisingly, as can be seen in FIG. 4,
the
addition of both a functional stf gene and a DNA payload that is packaged at
higher titers
due to its length drastically increases the delivery efficiency in vivo.
[217] Since strong differences were observed depending on the size of the
DNA
payload used, a set of experiments were performed where DNA payload of
different sizes
of SEQ ID NO: 4 to 8 were packaged into lambda capsids delivery vehicles and
their
titers assessed before the in vivo assays. The production was carried out in
the same
conditions for all payloads. After production and lysis following the protocol
mentioned
above, a step of TFF filtration with a 100 kDa pore size was carried out on
the cleared
lysates, and buffer exchanged for 1X PBS. The TFF step allows for the
concentration of
the particles about 1 log compared to the cleared lysates. As can be seen in
FIG.5 smaller
DNA payloads (2.2 kb) are produced at titers that are at least one log lower
than their
larger counterparts in our production system.
[218] Finally, to verify the roles of DNA payload size, titers and STF
presence, the 6
kb and 8 kb variants (GG6K of SEQ ID NO: 6 and GG8K of SEQ ID NO: 7,
respectively) shown in FIG. 5 were packaged in two lambda phagemid versions:
lambda
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
87
particles containing an intact full STF with its chaperone protein and lambda
particles
produced in a strain where the stf and tfa chaperone genes were seamlessly
deleted from
the prophage genome. Both versions encode the wild type lambda gpJ gene of SEQ
ID
NO: 10. After clearing, the lysates were passed through a TFF device with a
100 kDa
filter as described for the phagemids in FIG. 5 and their titers measured
(about 108/11L).
1010 total packaged phagemids of each DNA payload were gavaged to mice and the
delivery efficiency calculated as described above. Strikingly, as can be seen
in FIG. 6A,
the addition of a functional STF greatly increases the delivery efficiency in
vivo, which in
some cases is 100%.
[219] Significantly, these results highlight the importance of a functional
STF in in
vivo conditions, which does not reflect the results obtained in vitro, where
no obvious
difference can be observed using lambda Papa or Ur-lambda packaged phagemids
by
plating.
[220] This set of experiments brings three conclusions that have a major
impact for
the development of a successful approach to use packaged phagemids in vivo,
especially
for decolonization purposes. First, a DNA payload with a proper size to obtain
high
enough titers is necessary. If the DNA payload is too small, an intensive
downstream
processing focused on concentration of the particles to suitable titers must
be put in place,
which may slow down the production process and increase its cost. Second, the
presence
of a functional STF is essential to obtain the high delivery efficiencies
needed for in vivo
experiments, and it has been previously shown that STF can be engineered for
each target
strain (see for instance US provisional application US 62/802777, US
application
16/696,769 and US application 16/726,033 each of which is incorporated by
reference in
their entirety). Third, the primary receptor, LamB in this case, may not be
optimal for the
pursued application. As described below, it is shown that one can engineer
phagemid
particles to include gpJ variants (in the case of lambda) that bind other
receptors than
LamB.
Engineering the lambda gpJ protein
[221] The lambda phage uses the bacterial LamB OMP as its main entry
receptor
[15], which is recognized by the gpJ protein situated at the tip of the phage
particle. This
event triggers DNA ejection into the bacterial cytoplasm and is usually viewed
as an
"irreversible" binding process [15]. However, a bacterial strain can become
resistant to
lambda phage entry if the LamB receptor is mutated, masked or downregulated;
in
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
88
particular, downregulation of the LamB gene has been observed for MG1655
strains in
some mouse models, and this downregulation is caused by genotypic change: a
mutation
in the malT gene, regulating LamB expression levels, causes a drastic decrease
in the
number of these receptors in the membrane [16], [17]. In turn, bacteriophages
have
evolved different strategies to bypass these defense mechanisms. For instance,
mutating
the gpJ protein allows them to use a different receptor [18], [19]. It is also
known that the
receptor-recognition activity of gpJ lies in its C-terminal part, with a
fragment as small as
249 aa conferring the capability of binding to LamB receptors [5].
[222] Therefore, having different combinations of STF and gpJ that allow
entry in
specific conditions or different bacterial strains is crucial for the
successful utilization of
lambda-derived packaged phagemids as therapeutic agents, especially for in
vivo
applications. For this reason, several gpJ chimeras have been engineered that
recognize
receptors other than LamB, as well as identified variants that allow entry in
some E. coli
strains but not others.
[223] To achieve this, gpJ homologs from different E. coli prophage genomes
were
searched and a protein alignment to find the areas in the C-terminal part that
may be
involved in recognizing the bacterial OMP receptor was performed (FIG. 7).
[224] As shown in Figure 7 all gpJ variants share high sequence identity in
the
majority of their length (up to about amino acid 820) except for their C-
termini. To verify
that the receptor specificity is contained within this area of the gpJ
protein, different
fusions were tested using two insertion points (SEQ ID NO: 42 and SEQ ID NO:
43) as
shown in Figure 7. These chimeras share the N-terminus of the lambda gpJ and
differ in
their C-termini, which comes from other E. coli prophages. The gpJ variants
were
seamlessly inserted into the lambda production strain and packaged phagemids
containing
DNA payload p7.3kb (encoding a GFP and a chloramphenicol resistance gene) of
SEQ
ID NO: 9 produced as described above. Four strains were used to titrate these
packaged
phagemids: MG-GFP (a variant of MG1655 that encodes a GFP in the genome), MG-
delta-LamB (KEIO variant lacking the LamB receptor), H10-waaJ, a 0157 strain
lacking
the waaJ gene, which prevents the expression of the 0157 capsular antigen
[20]) and
MG1656-OmpC0157, a modified MG1655 in which the original OmpC has been
exchanged for the OmpC variant found in 0157 strains. The apparent titers
shown in
FIG. 8 are used as a measure of the efficiency of the packaged phagemids,
since the
0D600 of all strains was kept constant (0.8). As can be seen in FIG. 8., both
insertion
points yield functional gpJ chimeras, but surprisingly the recognized receptor
has changed
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
89
from the one recognized originally by lambda gpJ; for instance, as shown in
FIG. 8A, the
variant H591 of SEQ ID NO: 11 now uses OmpC. Interestingly, two of the gpJ
variants
(Z2145 of amino acid sequence SEQ ID NO: 12 and 1A2 of amino acid sequence SEQ
ID
NO: 13), show reduced or virtually no entry in MG1655, respectively, while
they are able
to recognize a receptor present in 0157 strains (FIG. 8). Neither variant uses
the LamB
receptor, as the titers in the MG-GFP and MG-delta-LamB strains are the same
(FIG.
8B). Finally, another gpJ variant was constructed and named A8 (SEQ ID NO: 49)
and its
delivery efficiency was tested on both MG1655 (containing its endogenous OmpC
variant) and MG1656-OmpC0157. To do this, serial 1:3 dilutions of phagemids
containing the A8 or 1A2 gpJ variants and a P2-stf chimera (protein of
sequence SEQ ID
NO: 50, typically encoded by the nucleic acid sequence SEQ ID NO: 56) were
incubated
with a fixed amount of MG1655 or MG1656-OmpC0157 cells at an 0D600 = 0.025; by
doing this, different MOIs were obtained. After incubation at 37 C for 45 min,
the GFP
levels of each MOI were measured using a flow cytometer and plotted against
the MOI.
As was observed previously, the 1A2 gpJ variant was only able to recognize the
receptor
in MG1656-OmpC0157; but surprisingly, the A8 variant was able to recognize
both the
OmpC receptor in MG1655 and MG1656-OmpC0157.
[225] In the light of these results, it seems that the approach of
generating gpJ
chimeras can produce not only variants that recognize different OMPs in the
same strain,
but also variants that show different strain specificity.
[226] However, changing the primary receptor is a futile effort if the
strain is
encapsulated: since the packaged phagemid will not have access to the receptor
due to a
physical masking of the cell surface, the delivery efficiency will be
drastically reduced. It
is for this reason that for the activity tests of the gpJ fusions shown above,
the naked H10-
waaJ 0157 strain was used, since it is known that 0157 strains produce a group
IV
capsular antigen that masks the cellular surface [21]. In this case, a
combination of a
functional STF and gpJ is necessary to obtain high delivery efficiencies.
[227] To do this, stf and the tfa (chaperone) genes from the lambda
prophage in the
production strain were seamlessly deleted. Chimeric stfs were then
complemented in
trans with a plasmid carrying DAPG-inducible versions of these chimeric stfs.
Indeed,
when using a STF variant referred to as WW11.2 and represented by SEQ ID NO:
16 that
shows specificity for the 0157 antigen, in combination with the gpJ variant
Z2145 of
amino acid sequence SEQ ID NO: 12 shown above, very efficient entry in
encapsulated
0157 strain were obtained, as can be seen in FIG. 8C.
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
Engineering the lambda tape measure protein, gpH
[228] Additionally, it has been shown that another possible mechanism for
bacteria
to protect from phage injection is the mutation of periplasmic proteins that
are believed to
assist in the formation of a channel across the periplasm while the DNA is
being injected
[22]424]. Lambda phage can regain its activity in these mutants by modifying
the gpH
gene, the tape measure protein, although the exact position of these mutations
is not
known. In the lambda phage, the protein involved in the interaction with the
mannose
permease complex is gpH. Recently, it has been described that some E. coli
strains
become resistant to infection by HK97, a lambdoid phage, by mutations in the
glucose
transporter protein PtsG; the gpH protein of HK97 is inhibited and injection
into these
mutants cannot occur. The authors describe that by changing a region of the
gpH protein
in the HK97 phage they could bypass the PtsG mutation and the engineered HK97
becomes infectious again.
[229] To test if this hypothesis is generalizable to the lambda phage,
alignments of
the lambda gpH protein to other lambdoid prophages present in E. coli was
performed. As
shown in FIG. 9A, other variants of gpH can be found where there is an
extremely high
percentage of identity across the length of gpH except for two regions
(labeled distal and
proximal) which show heterogeneity in their sequence.
[230] The gpH gene was modified in the lambda packaged phagemid production
strain, as was done for the gpJ variants, to include both variable regions and
packaged
phagemids were produced as described above. This gpH variant of SEQ ID NO: 24
was
termed gpH-IAI. In this case, three strains were used for titration: MG1655,
KEIO manY
and KEIO manZ, which contain deletions of two components of the mannose
permease
complex. As can be seen in FIG. 9B, the deletion of manZ and manY causes a 2-
log
decrease in the apparent titer as compared to MG1655; however, using the
modified gpH
lambda phage variant of SEQ ID NO: 24 fully restores its activity, even in the
manZ and
manY strains. These results show that modifying the gpH gene in lambda phage
can be
used to allow or improve entry in strains that show deficiencies or changes in
the
permease complexes.
[231] With this battery of experiments, it is shown that one is able to
engineer the
three main mechanisms involved in lambdoid phage recognition and injection
into its
host: capsule/secondary receptor (through STF); primary receptor recognition
(through
gpJ); and permease/periplasmic channel formation (through gpH). These
modifications
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
91
can be used to engineer lambdoid phages and packaged phagemids with modified
tropism
and injection efficiency in a wide number of E. coli strains.
Using lambda phagemids to efficiently deliver to different, unmodified
Proteobacteria
[232] Finally, it has been shown that lambdoid-derived packaged phagemids
can be
used to deliver in other Proteobacteria different from E. coli, such as
Klebsiella,
Agrobacterium, or Pseudomonas. [25]427]. However, this approach requires the
transformation of the receiver strains with a plasmid encoding the E. coli
LamB receptor,
since other species do not possess it. While this is a valid approach for
cells grown in
laboratory conditions, it becomes infeasible if bacteria present in natural
conditions are to
be targeted with lambda-derived packaged phagemids (for instance, the gut),
since the
plasmid encoding the receptor cannot be easily transferred.
[233] It was tested if the lambda-derived packaged phagemids could be used
to
deliver the p7.3 kb payload of SEQ ID NO: 9 to other Proteobacteria, such as
Enterobacter. To achieve this, different lambda packaged phagemids were
engineered to
contain several combinations of gpJ, Z2145 of SEQ ID NO: 12 and 1A2 of SEQ ID
NO:
13 and, STF variants STF-EB6 of SEQ ID NO: 19 (with its chaperone (or
accessory)
protein of SEQ ID NO: 20), 5TF75 of SEQ ID NO: 17 (with its accessory protein
of SEQ
ID NO: 18) and 5TF23 of SEQ ID NO: 21 (with its accessory protein of SEQ ID
NO:
22). FIG. 10 shows that the same principles hold as observed for E. coli
strains: the
delivery efficiency depends strongly on the choice of gpJ and STF used; for
some
combinations, entry in these bacteria is inefficient (although transductants
can be readily
seen) but changing the STF and gpJ allows for a much higher delivery
efficiency.
[234] These results show that injection elements of the lambda capsid (gpJ,
stf, gpH)
can be engineered to achieve high delivery efficiency in other type of
bacteria than E.
coli. Accordingly, the present inventors showed that it is advantageous to use
one or more
of these approaches (DNA payload size optimization and selection of a proper
gpJ/STF/gpH combination) to generate optimized lambdoid-based delivery
vehicles to be
used in in vivo experiments involving transfer of DNA.
EXAMPLE 2
Using lambda phagemids to efficiently deliver in bacteria in vivo
[235] The inventors have tested if a payload inside packaged phagemids with
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
92
lambda STF fusions based on homology points could be delivered to bacteria in
the
gastrointestinal tract of a host without such STF being affected by
proteolytic activity
present in the gastrointestinal tract, in particular by pancreatin (i.e. low
to no delivery of
the payload to bacteria)
[236] In principle, the structure of homology-based STF chimeras should be
reminiscent of the original proteins, as the amino acid sequence of the fusion
points has
not been majorly modified. This means that if the original STFs had evolved to
be
pancreatin resistant, it is quite probable that the chimera will also be
resistant. To prove
this, the inventors have engineered two different STF chimeras with lambda STF
functional when targeting LMR 503 strain (lambda-STF29, SEQ ID NO: 63 with its
accessory protein of sequence SEQ ID NO: 65; and lambda-STF118, SEQ ID NO: 64
with its accessory protein of sequence SEQ ID NO: 66). The insertion point of
5TF29 is
ADAKKS (SEQ ID NO: 38) and the insertion point of STF118 is MDETNR (SEQ ID
NO: 39). Eligobiotics harboring the 1A2 gpJ and each of the STF chimeras were
produced and titrated on MG1656-OmpC0157 or LMR 503 after treatment with or
without pancreatin at pH 6.8. Briefly, the readout strain for chimeric STF
activity is
LMR 503 and the readout for gpJ activity is MG1656-OmpC0157. As can be seen in
FIG. 11, these STF chimeras based on homology points show virtually no
degradation in
the presence of pancreatin, as predicted by the inventors based on STF
chimeras
homology-designed fusion points.
EXAMPLE 3
Effect of DNA payload size on Eligobiotics packaging
[237] To evaluate the effect of DNA payload size on the number of payloads
packaged in Eligobiotics (EB), 3 different payloads were used to produce
Eligobiotics
as summarized in Table 1.
Table 1: Batches of Eligobiotics produced
Eligobiotic code / batch number Payload name Size (kb)
eb512 / EB003-DS-008 p1085 12.125
eb393 / EB003-DS-009 p779 12.428
eb827 / EB003-DS-011 p1392 11.615
[238] After fermentation, lysis (3 h incubation at 37 C with 0.1% Triton X-
100,
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
93
2000 U/L Benzonase) and clarification on a Zeta Plus Capsule (3M),
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.
[239] Analysis of Eligobiotics 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 EBs present in solution for each
EB batch
were extracted from sedimentation velocity data (acquired at 260 and 280 nm).
[240] Based on the molecular weight calculated from their sedimentation
coefficient
and their 260/280 nm ratios, the different populations of EBs 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 (FIG. 12).
[241] 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 EBs
contained 4
copies of the payload (FIG. 13).
[242] Thus, it appears that p1392 is close to an ideal size to package
exclusively 4
copies of payload in phage-derived capsids, yielding an homogenous population.
Increasing the size of the payload compared to p1392 generates more
heterogeneous
Eligobiotics populations, with increasing proportions of EBs 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 FIG. 13. 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
CA 03164131 2022-06-09
WO 2021/136812 94
PCT/EP2020/088043
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).
[243] From these data, the inventors can also predict 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.
Table 2: Predicted number of concatemers packaged in a capsid depending on the
monomer size.
Number of copies in the concatemer
Plasmid size (kb) 2 3 4 5 6 7 8 9
10 11
3 6 9 12 15 18 21 24 27 30 33
4 8 12 16 20 24 28 32 36 40 44
10 15 20 25 3111 35 40 45 50 55
6 12 18 24 3C 76 4? 42 54 F0 F6.
7 14 21 28 35 42 4g 56 63 70 77
16 24 32 io is 56 64 72 80 88
9 18 27 ?,6 45 54 63 72 81 90 99
20 30 41: 0 fL1 70 80 90 100 110
Single conformation possible, 4 copies 11 22 33 44 55 66
77 88 99 110 121
12 24 36 42 CO 72 84 96 108 120 132
Single conformation possible 13 26 39 'D2 b.5 78
91 104 117 130 143
Single conformation possible 14 2S 42 56 70
84 98 112 126 140 154
Single conformation possible 15 30 45 6C: 75
90 105 120 135 150 165
Single conformation possible 16 72 4 64 80
96 112 128 144 160 176
Sing ie conformation possible, high limit 17 74 51 68 85
102 119 136 153 170 187
Single conformation possible 18 76 51 72
90 108 126 144 162 180 198
Single conformation possible 19 33 57 76
95 114 133 152 171 190 209
Single conformation possible 20 40 GO 80
100 120 140 160 180 200 220
Single conformation possible 21 42 61 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
Shadowed cells represent better species, white cells 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.
SUBSTITUTE SHEET (RULE 26)
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
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
10 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 43 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
Shadowed cells represent better species, white cells 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.
SEQUENCES
Name SEQ ID NO: Type
psgRNAcos (p184) 1 DNA
pJ23104-GFP (p211) 2 DNA
pJF1 (p344) 3 DNA
GG2K (p502) 4 DNA
GG4K (p'710) 5 DNA
GG6K (p504) 6 DNA
GG8K (p711) 7 DNA
GG12K (p499) 8 DNA
p7.3kb (p513) 9 DNA
Lambda gpJ (Uniprot P03749) 10 Amino acid
gpJ 591 11 Amino acid
gpJ Z2145 12 Amino acid
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
96
1A2 13 Amino acid
Lambda STF (Uniprot P03764) 14 Amino acid
Lambda tfa (Uniprot P03740) 15 Amino acid
STF lambda-WW11.2 16 Amino acid
STF lambda-75 17 Amino acid
Accessory protein lambda-STF75 18 Amino acid
STF lambda-EB6 19 Amino acid
Accessory protein lambda-EB6 20 Amino acid
STF lambda-23 21 Amino acid
Accessory protein lambda-STF23 22 Amino acid
Lambda gpH (Uniprot P03736) 23 Amino acid
gpH-JAI 24 Amino acid
H591 25 DNA
Z2145 26 DNA
1A2 27 DNA
Lambda stf 28 DNA
Lambda tfa 29 DNA
Stf lambda-WW11.2 30 DNA
Stf lambda-75 31 DNA
Accessory protein lambda-stf75 32 DNA
Stf lambda-EB6 33 DNA
Accessory protein lambda-EB6 34 DNA
Stf lambda-23 35 DNA
Accessory protein lambda-stf23 36 DNA
Insertion site SAGDAS 37 Amino acid
Insertion site ADAKKS 38 Amino acid
Insertion site MDETNR 39 Amino acid
Insertion site SASAAA 40 Amino acid
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
97
Insertion site GAGENS 41 Amino acid
Insertion point 1 Figure 7 42 Amino acid
Insertion point 2 Figure 7 43 Amino acid
STF-V10 44 Amino acid
STF-V1Of 45 Amino acid
STF-V10a 46 Amino acid
Example of payload sequence 47 DNA
STF-V10h 48 Amino acid
A8 49 Amino acid
STF lambda-P2 50 Amino acid
stf-V10 51 DNA
stf-V10f 52 DNA
stf-V10a 53 DNA
stf-V10h 54 DNA
A8 55 DNA
Stf lambda-P2 56 DNA
P2 accessory protein 1 57 Protein
P2 accessory protein 1 58 DNA
Sequence of Z2145 gpJ of Figure 7 59 Protein
Sequence of 1A2 gpJ of Figure 7 60 Protein
Sequence of 591 gpJ of Figure 7 61 Protein
Sequence of E6BTD4-ECOLX gpH of 62 Protein
Figure 9A
Lambda-5TF29 63 Protein
Lambda-STF118 64 Protein
Lambda-5TF29 accessory protein 65 Protein
Lambda-STF118 accessory protein 66 Protein
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
98
REFERENCES
[1] J. Collins and B. Hohn, "Cosmids: a type of plasmid gene-cloning vector
that is
packageable in vitro in bacteriophage lambda heads.," Proc. Natl. Acad. ScL U.
S. A.,
vol. 75, no. 9, pp. 4242-4246, Sep. 1978.
[2] J. E. Cronan, "Cosmid-Based System for Transient Expression and Absolute
Off-
to-On Transcriptional Control of Escherichia coli Genes,"J. BacterioL, vol.
185, no. 22,
pp. 6522-6529, Nov. 2003, doi: 10.1128/JB.185.22.6522-6529.2003.
[3] J. E. Cronan, "Improved Plasmid-Based System for Fully Regulated Off-To-On
Gene Expression in Escherichia coli: Application to Production of Toxic
Proteins,"
Plasmid, vol. 69, no. 1, pp. 81-89, Jan. 2013, doi:
10.1016/j.plasmid.2012.09.003.
[4] J. D. Haley, "Cosmid library construction," Methods MoL BioL Clifton NJ,
vol. 4, pp.
257-283, 1988, doi: 10.1385/0-89603-127-6:257.
[5] J. Wang, M. Hofnung, and A. Charbit, "The C-terminal portion of the tail
fiber
protein of bacteriophage lambda is responsible for binding to LamB, its
receptor at
the surface of Escherichia coli K-12,"J. BacterioL, vol. 182, no. 2, pp. 508-
512, Jan.
2000, doi: 10.1128/jb.182.2.508-512.2000.
[6] J. H. Grose and S. R. Casj ens, "Understanding the enormous diversity of
bacteriophages: the tailed phages that infect the bacterial family
Enterobacteriaceae," Virology, vol. 0, pp. 421-443, Nov. 2014, doi:
10.1016/j.viro1.2014.08.024.
[7] R. W. Hendrix and R. L. Duda, "Bacteriophage lambda PaPa: not the mother
of all
lambda phages," Science, vol. 258, no. 5085, pp. 1145-1148, Nov. 1992.
[8] M. G. Rossmann, V. V. Mesyanzhinov, F. Arisaka, and P. G. Leiman, "The
bacteriophage T4 DNA injection machine," Curr. Opin. Struct. BioL, vol. 14,
no. 2, pp.
171-180, Apr. 2004, doi: 10.1016/j.sbi.2004.02.001.
[9] Y. Zivanovic et al., "Insights into Bacteriophage TS Structure from
Analysis of Its
Morphogenesis Genes and Protein Components,"/ ViroL, vol. 88, no. 2, pp. 1162-
1174, Jan. 2014, doi: 10.1128/JVI.02262-13.
[10] M. A. Speed, T. Morshead, D. I. Wang, and J. King, "Conformation of
P22
tailspike folding and aggregation intermediates probed by monoclonal
antibodies,"
Protein ScL PubL Protein Soc., vol. 6, no. 1, pp. 99-108, Jan. 1997, doi:
10.1002/pro.5560060111.
CA 03164131 2022-06-09
WO 2021/136812 PCT/EP2020/088043
99
[11] T. Miwa and K. Matsubara, "Formation of oligomeric structures from
plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda
heads.,"J. BacterioL, vol. 153, no. 1, pp. 100-108, Jan. 1983.
[12] P. Grayson et aL, "The effect of genome length on ejection forces in
bacteriophage lambda," Virology, vol. 348, no. 2, pp. 430-436, May 2006, doi:
10.1016/j.viro1.2006.01.003.
[13] S. Vimont et aL, "The CTX-M-15-producing Escherichia coli clone 025b:
H4-ST131 has high intestine colonization and urinary tract infection
abilities," PloS
One, vol. 7, no. 9, p. e46547, 2012, doi: 10.1371/journal.pone.0046547.
[14] M. L. Myhal, D. C. Laux, and P. S. Cohen, "Relative colonizing
abilities of
human fecal and K 12 strains of Escherichia coli in the large intestines of
streptomycin-treated mice," Eur. J. Clin. MicrobioL, vol. 1, no. 3, pp. 186-
192, Jun.
1982.
[15] S. Chatterjee and E. Rothenberg, "Interaction of Bacteriophage A. with
Its E.
coli Receptor, LamB," Viruses, vol. 4, no. 11, pp. 3162-3178, Nov. 2012, doi:
10.3390/v4113162.
[16] A. Charbit, K. Gehring, H. Nikaido, T. Ferenci, and M. Hofnung,
"Maltose
transport and starch binding in phage-resistant point mutants of maltoporin.
Functional and topological implications,"J. MoL BioL, vol. 201, no. 3, pp. 487-
496,
Jun. 1988.
[17] M. D. Paepe, V. Gaboriau-Routhiau, D. Rainteau, S. Rakotobe, F.
Taddei,
and N. Cerf-Bensussan, "Trade-Off between Bile Resistance and Nutritional
Competence Drives Escherichia coli Diversification in the Mouse Gut," PLOS
Genet.,
vol. 7, no. 6, p. e1002107, Jun. 2011, doi: 10.1371/journal.pgen.1002107.
[18] J. R. Meyer, D. T. Dobias, J. S. Weitz, J. E. Barrick, R. T. Quick,
and R. E.
Lenski, "Repeatability and contingency in the evolution of a key innovation in
phage
lambda," Science, vol. 335, no. 6067, pp. 428-432, Jan. 2012, doi:
10.1126/science.1214449.
[19] C. Werts, V. Michel, M. Hofnung, and A. Charbit, "Adsorption of
bacteriophage lambda on the LamB protein of Escherichia coli K-12: point
mutations
in gene J of lambda responsible for extended host range,"J. BacterioL, vol.
176, no. 4,
pp. 941-947, Feb. 1994, doi: 10.1128/jb.176.4.941-947.1994.
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
100
[20] K. S. Choi etal., "Protection from Hemolytic Uremic Syndrome by
Eyedrop
Vaccination with Modified Enterohemorrhagic E. coli Outer Membrane Vesicles,"
PLoS ONE, vol. 9, no. 7, Jul. 2014, doi: 10.1371/journal.pone.0100229.
[21] Y. Shifrin et al., "Transient Shielding of Intimin and the Type III
Secretion
System of Enterohemorrhagic and Enteropathogenic Escherichia coli by a Group 4
Capsule,"/ BacterioL, vol. 190, no. 14, pp. 5063-5074, Jul. 2008, doi:
10.1128/JB.00440-08.
[22] C. A. Roessner and G. M. Ihler, "Proteinase sensitivity of
bacteriophage
lambda tail proteins gpJ and pH in complexes with the lambda receptor.,"J.
BacterioL, vol. 157, no. 1, pp. 165-170, Jan. 1984.
[23] D. Scandella and W. Arber, "Phage lambda DNA injection into
Escherichia
coli pel- mutants is restored by mutations in phage genes V or H," Virology,
vol. 69,
no. 1, pp. 206-215, Jan. 1976.
[24] I. Martin-Verstraete, V. Michel, and A. Charbit, "The levanase operon
of
Bacillus subtilis expressed in Escherichia coli can substitute for the mannose
permease in mannose uptake and bacteriophage lambda infection,"J. BacterioL,
vol.
178, no. 24, pp. 7112-7119, Dec. 1996, doi: 10.1128/jb.178.24.7112-7119.1996.
[25] G. E. de Vries, C. K. Raymond, and R. A. Ludwig, "Extension of
bacteriophage lambda host range: selection, cloning, and characterization of a
constitutive lambda receptor gene," Proc. Natl. Acad. ScL U. S. A., vol. 81,
no. 19, pp.
6080-6084, Oct. 1984.
[26] M. S. Francis, A. F. Parker, R. Morona, and C. J. Thomas,
"Bacteriophage
Lambda as a Delivery Vector for Tn10-Derived Transposons in Xenorhabdus
bovienii," App!. Environ. MicrobioL, vol. 59, no. 9, pp. 3050-3055, Sep. 1993.
[27] R. A. Ludwig, "Gene tandem-mediated selection of coliphage A.-
receptive
Agrobacterium, Pseudomonas, and Rhizobium strains," Proc. Natl. Acad. ScL U.
S. A.,
vol. 84, no. 10, pp. 3334-3338, May 1987.
[28] T. Miwa and K. Matsubara, "Formation of oligomeric structures from
plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda
heads." J Bacteriol. vol. 153, no. 1, pp. 100-108, Jan 1983.
[29] I. Ivanovska, G. Wuite, B. JOnsson, and A. Evilevitch, "Internal DNA
pressure modifies stability of WT phage" Proc. Natl. Acad. Sci. U. S. A., vol.
104, no.
23, pp. 9603-9608, June 2007.
CA 03164131 2022-06-09
WO 2021/136812
PCT/EP2020/088043
101
[30] E.
Nurmemmedov, M. Castelnovo, E. Medina, C. Enrique Catalano and A.
Evilevitch, "Challenging packaging limits and infectivity of phage X" J. Mol.
Biol. vol.
415, no. 2, pp. 263-73, Jan. 2012
