WO2011080505A2

WO2011080505A2 - Method of growing phage for commercial use

Info

Publication number: WO2011080505A2
Application number: PCT/GB2010/002341
Authority: WO
Inventors: John Bernard March; Jason Richard Clark
Original assignee: Bigdna Ltd
Priority date: 2009-12-28
Filing date: 2010-12-24
Publication date: 2011-07-07
Also published as: WO2011080505A3

Abstract

The present invention relates to methods of obtaining bacteriophage ('phage'), such as lambda phage, for industrial uses, such as for use in commercial vaccine production. There is also provided a novel bacterium, such as Escherichia coli (E. coli) which has been modified for growing phage in a manner which improves the general and environmental safety of said phage. The invention also relates to the use of a modified bacterium for growing phage for industrial purposes. Finally, the invention also provides modified phage, which are designed for growth in industrial and/or environmental safety.

Description

Method of Growing Phage for Commercial Use

Field of Invention

The present invention relates to methods of obtaining bacteriophage ('phage'), such as lambda phage, for industrial uses, such as for use in commercial vaccine production. There is also provided a novel bacterium, such as Escherichia coli (£. coli) which has been modified for growing phage in a manner which improves the general and environmental safety of said phage. The invention also relates to the use of a modified bacterium for growing phage for industrial purposes. Finally, the invention also provides modified phage, which are designed for growth in industrial and/or environmental safety.

Background to the Invention

WO02/076498 teaches the use of bacteriophage to express an immunogenic protein/peptide, in order to develop immunogenic compositions and DNA vaccines. Other patents, such as WO9951780 and WO05058006, also discuss using phage as vectors for delivering immunogenic peptides.

One particularly suitable vector is phage lambda and its derivatives, due to their relatively well characterised biology, handling ability and cloning characteristics. However it is to be understood that any bacteriophage may be used to practice the invention as described herein. However, if such phage are to be used for industrial application, such as in medical or biotechnological processes, there are a number of crucial parameters which must be addressed. Firstly, appropriate methods and processes for production of sufficient quantities of the phage will be required, which requires growth on a suitable bacterial host. Secondly, there are regulatory concerns about the release of genetically modified material into the environment and so appropriate safety considerations for the phage vector and the methods by which it is produced, need to be addressed. Finally, it will be a regulatory requirement of the industrial scale process to ensure the consistency of the phage DNA or phage particles produced and so such aspects must be addressed in the design of the phage vector and the manner by which the end products are manufactured. WO2003/070880 and WO2007/024756 describe the development of reduced genome E. coli strains to improve the production of recombinant proteins, nucleic acids and amino acids but there is no suggestion of using such reduced genome E.colito improve phage production, which may include either phage DNA or the whole phage particles. Moreover, the E.coli described contain a range of deletions/modifications which are both unnecessary and inappropriate for phage production. Furthermore, and importantly, the E. coli described may not include deletions/modifications deemed most appropriate for improving growth of phage, nor address the safety and regulatory issues critical to development of an industrially applicable process for manufacture of a product destined for medical use.

It is therefore amongst the objectives of the present invention to obviate and/or mitigate one or more of the aforementioned issues. It is an objective of the present invention to provide a process for preparing large quantities of phage for industrial purposes. It is a further objective of the present invention to provide a phage, such as a lambda phage that is capable of being grown in the laboratory in large scale, whilst having a reduced capacity for infection/replication in the wild. It is a further objective of the present invention to provide a phage and a bacterial host cell to be used in a process which is capable of achieving and maintaining a satisfactory degree of consistency of phage product when grown at industrial scale.

Summary of the Invention

In a first aspect there is provided a method of method of obtaining bacteriophage for commercial purposes, the method comprising:

(a) growing a bacterium which is deficient in one or more Type I restriction and/or modification genes and/or deficient for one or more genes ecoding for fimbriae and/or flagella protein; and

(b) infecting said bacterium with a suitable phage in order to allow multiplication of said phage; or inducing growth of a prophage which has previously been integrated into the genome of the bacterium, in order to allow multiplication of phage.

In a further aspect there is provided use of a bacterium which is deficient in one or more restriction and/or modification genes for growing phage and/or deleted for genes such as the fimbriae and flagella. It is expected that rendering deficient one or more restriction and/or modification genes will allow for phage to be grown in the modified bacterium, but which are able to grown in wild-type bacteria less efficiently. Also rendering deficient one or more flagella and/or fimbrae genes may allow for easier purification of phage so produced.

Commercial purposes include (but are not restricted to) the use of phage in the production of immunogenic compositions and/or vaccines, and for gene therapy applications. Other applications could include using phages to treat bacterial infections, using phages for the purposes of bacterial decontamination, using phages to detect and/or type bacteria or where phages are produced with modified coat proteins, or have their coat proteins modified post- production, for biotechnological purposes and where it is desirable to restrict the growth of such phages in the environment.

The present invention is applicable to any bacterium which possesses a Type I restriction and/or modification system and includes £. coli, Salmonella species, such as Salmonella typhimurium (S. typhi), Shigella sp., such as Shigella flexneri, various entercocci sp. such as Klebsiella, Proteobacteria, such as Psuedomonas sp. and Haemophilus sp. and Firmicutes bacteria, including Bacillus sp. and Steptococcus sp. E. coli and S. typhi are preferred.

Any phage such as a dsDNA, or ssDNA, ssRNA, and dsRNA phage which is capable of infecting one or more of the aforementioned bacteria, may be employed, but a preferred phage type is a dsDNA phage, e.g. a lamboid phage such as phage lambda. However, other dsDNA phage may also be employed such as T phages (including T2-T7), and ssDNA phage such as M13.

Although it is not to be construed as limiting, for ease of reference, the remainder of this description will generally refer to E. coli and lamboid phage and the skilled reader will be aware of equivalents which are of relevance to bacteria other than E.coli and phage other than lamboid phage.

Said one or more Type I restriction, and/or modification genes to be rendered deficient in an E. coli of the present invention are exemplified by the hsdSMR genes of E. coli K12 but may also include one or more of the other restriction modification systems of E. coli as exemplified by the mcr and mrr gene families. Preferably all known nucleic acid restriction and modification genes/proteins are rendered deficient in the E. coli according to the present invention.

Said one or more genes associated with fimbriae and/or flagella production, which may be rendered deficient may include flagellin structural proteins, ygll (a putative fimbrial like protein, flgA-H (genes associated with flagellin biosynthesis); flhA-E (genes associated with flagellin synthesis/chemotaxis; /7/A-R (genes associated with flagellin biosynthesis); fimS- (genes associated with fimbriae biosynthesis).

By "deficient" it is meant that the E.coli is substantially incapable of expressing said particular gene(s) in a functionally active form. This may be achieved, for example, by partial or complete deletion of a particular sequence, by appropriate mutation, or by an insertion into the gene(s) or inversion of the sequence, in order to render the gene(s) incapable of expressing a functionally active form of the encoded protein.

Optionally the E.coli of the present invention may be further modified to lack nucleic acid sequence(s) which is/are homologous with the infecting phage. The lack of nucleic acid which is homologous with the infecting phage is designed to prevent recombination of the phage with the host bacterium's genome which could result in genetic material from the host being incorporated into the genomes of progeny phage, or integration of genetic material from the phage being incorporated into the host. Ideally, no regions of homology of greater than 20 or 25 bp will be found between the phage being produced and the host bacterium. It will be understood that when a prophage sequence is found within a bacterium's genome, not all sequences of the prophage need to be deleted. For example, only the sequences of the prophage which are homologous with the infecting phage (or phages), may be deleted, or portions thereof, so as to minimise recombination.

The skilled addressee is aware of many techniques to render the E. coli deficient, or lack particular sequences, these include techniques designed to delete the whole or part of a particular sequence, as well as mutagenesis techniques which may introduce mutation through substitution, for example, introducing a stop codon; inversion and transversion. Exemplary techniques for producing genome deletions in bacteria, such as E. coli are described for example in Mizoguchi ef a/., 2007, Wang at al., 2006 and Posfai ef al., 2006 and Sambrook ef al., 2001. A preferred E. coli for use as a parent stain (i.e prior to rendering deficient for said one or more genes) is the strain MG1655.

MG1655 was sequenced by the Blattner laboratory (Blattner et al 1997 Science, 277 (5331); P1453-62 Gene Bank Accession No. U00096). The mutations listed in the strain genotype are present in most K-12 strains and were probably acquired early in the history of the laboratory strain. A frameshift at the end of the /? results in decreased pyrE expression and a mild pyrimidine starvation, such that the strain grows 10 to 15 % more slowly in pyrimidine-free medium than in a medium containing uracil. The /M3-mutation is a frame shift that knocks out acetohydroxy acid synthase II. The rfb-50 mutation is an IS5 insertion that results in the absence of O-antigen synthesis.

In addition to the requirements of a deficiency of one or more genes associated with restriction and/or modification of DNA and/or deficient for one or more genes ecoding for fimbriae and/or flagella protein(s), further deletions/mutations from within the E. coli genome may be beneficial to enhance phage production, in terms of increased or more efficient growth of a phage and/or improved ability of purifying the phage so produced and/or enhance safety. Thus, the E. coli of the present invention may further comprise a deletion and/or mutation in/of one or more of the following nucleic acid sequences. fhuA - this encodes the phage T1 receptor. Removal of fhuB-D may also be carried out, but may not be necessary following fhuA deletion/mutation;

Deletion/mutation of one or more prophage elements, such as a rac (a lamboid prohophage present in the E. coli k12 genome); Qin (which is also a prophage present in the E. coli k12 genome); inst B (from the P4 prophage; J4; e14; DLP12; and ogrK (a prophage P2 protein);

Deletion/mutation of one or more endonuclease genes such as encIA; Deletion/modification of one or more DNA repair genes, such as recA;

Deletion/modification of one or more toxic genes/proteins, such as hokA, hokC, hokE and mokC; and/or Deletion/modification of one or more genes associated with auto-aggregation, such as flu (antigen 43).

In addition the host bacteria may also be deleted for, or contain a modified phage binding protein. When phage infect bacteria, they must specifically bind to some surface exposed component of the bacterial cell, such as LPS or an outer membrane protein. An example of this is lamB, the maltose binding protein found in E. coli which is the binding target for bacteriophage lambda. The host cells described may contain a modified phage binding target, such that wild-type phages cannot infect the host cell. These modified hosts would be used in combination with a modified phage, which had been adapted (by selection) to grow on the modified host. Using such a bacterial host in a production environment would result in progeny phage, which were unable to infect wild-type bacteria and were therefore safe for release into the environment.

Many of the above deletions are described in more detail in WO07/024756 to which the skilled reader is directed and the contents of which are hereby incorporated in their entirety.

Although many deletions/mutations may be carried out in order to provide an E. coli which allows industrial growth of phage, for commercial purposes, it is preferable that more substantial deletions of the E. coli genome are not carried out, to preserve the functionality of the strain and for regulatory reasons (i.e to minimise the amount of genetic modification performed on the host). Thus, it is preferred that typically less than 4% such as 3.5%, 3%, 2.5%, 2%, 1.5% or 1% of the parent, such as ( G1655), E. coli genome (in terms of total kilobases) is deleted.

It is desirable to be able to grow phage on an industrial scale (i.e. in a fermentor or other bioreactor, such as a disposable vessel) so that large quantities of phage can be obtained for processing into a suitable product, which necessarily will be released into the environment, such as a vaccine or other biological product for administration to a subject; it is also highly desirable that the phage so produced are not readily able to infect wild-type bacteria, e.g. wild-type E. coli and to further replicate other than in the modified host bacterium of the present invention. Should the phage infect a wild-type E. coli it is expected that the phage genome will be restricted, due to the presence of said type-I restriction sites and/or lack of restriction modification altering proteins, thereby minimising/reducing growth of the phage in the wild, (i.e outside controlled laboratory phage production facilities).

In a further aspect there is provided a modified phage for growth in industrial scale, the phage comprising one or more restriction notification affecting proteins which have been rendered deficient; and/or one or more introduced type-l restriction sites, in order to reduce the ability of the phage to survive in the wild (i.e. other than growth in the modified laboratory bacterium of the present invention).

In accordance with the present invention it is possible to grow phage in high-titre in a desired laboratory bacterium, but wherein phage are able to grow far less efficiently in a "wild-type" bacterium (i.e. not the desired laboratory bacterium).

Thus, the phage used to infect a host bacterium, such as E. coli described herein, may comprise those genetically modified to include multiple copies of one or more type-l restriction sites (Figure 12 and Murray, 2000), such as at least 1 , 2,3, 4, 5, 6, 7, 8, 9, or 10 type-l restriction sites, in addition to those which occur naturally in the parental phage. Examples of such sites would include but are not limited to the recognition sequences for Eco377l, Eco394l, Eco585l, Eco646l, EC0777I, Eco826l, Eco851 l, Eco912l, EcoAI, EcoBI, EcoDI, EcoDXXI, EcoEI, EcoKI, EcoPrrl, EcoR124l, EcoR124ll, StyLTIII, StySBLI, StySEAI, StySENI, StySGI, StySJI, StySKI, StySPI and StySQI.

Such phage also preferentially lack one or more restriction modification affecting proteins, for example the restriction modification (ral) protein of bacteriophage lambda. The phage so described will thus inhibit the action of host restriction enzymes so that the phage is not degraded. However, should the phage infect a wild-type E. coli it is expected that the phage genome will be restricted, due to the presence of said type-l restriction sites and/or lack of restriction modification altering proteins, thereby minimising/reducing growth of the phage in the wild, (i.e outside controlled laboratory phage production facilities).

Further optionally mutations/deletions may be provided. Such one or more nucleotide sequence deletions may be from genes, whole or fragments thereof, deemed not to be required for industrial scale growth, and/or regulatory sequences, (e.g. promoters and/or terminators) which are not deemed necessary for industrial scale growth. It is to be appreciated that any deleted sequence, if not replaced by nucleic acid with a specific function may be replaced with a "stuffer" nucleic acid of either random or designed sequence with no biological function, so as to maintain at least the minmum genome size required to produce viable phage.

The phage genome may be modified by deletion of one or more portions of the genome which may serve to still allow/enable growth in the laboratory, such as a modified laboratory strain of the present invention, whilst reducing/minimising growth of the modified phage in the wild i.e. when not grown in the laboratory under controlled conditions. Examples of portions of a phage genome which may be deleted include portions/genes which may be responsible for lysogeny, certain promoters and/or terminators, and/or certain tail-fibre genes/promoters. Specific portions of the lambda phage genome (and homologous portions from genomes of other phage), which could be removed to increase safety and/or increase cloning capacity are identified in Figure 16. It will be appreciated that one or more of the identified sequences, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all identified sequences, may be deleted.

In addition to said Type I restriction sites, the phage of the present invention may also be engineered to possess one or more Type II restriction sites. Not only will these Type II sites within the phage serve to act as "cloning" sites to enable/facilitate cloning/genetic manipulation of exogenous sequences into the phage genome, but they will also act to provide a further level of safety as these sites will be able to be restricted, in a similar manner to the Type I sites, should the phage infect a bacterium other than a laboratory host strain. Of course, the laboratory host strain should not possess the restriction enzyme(s) corresponding to said Type II site which is introduced into the phage genome.

Additionally, the phage genome may be modified to include artificial type I restrictions sites. It is well established (for example Murray, 2000; Titheradge et al, 2001) that bacteria can generate new Type I restriction sites by recombination of the two separate domains which comprise the recognition site. To reduce the chances of replication in the environment, phages covered by this invention may be modified to include synthetic Type I restriction sites based on the sequences of sites already characterised (Figure 12 and Figure 14b).

The phage genome may also be modified by altering its nucleic acid sequences in order to minimise homologous recombination with the host genome. The skilled addressee is well aware that many codons can be replaced with alternate codons, which encode the same amino acid. In this manner, the phage genome can be engineered and/or synthesised in part or in whole, in order to utilise codons and hence stretches of nucleic acid which are not homologous with the host genome or prophage sequences present within it (but which encode for the same original protein sequence). One relatively easy method is to modify the third base of many codons as this can provide a codon which although different, still encodes the same amino acid.

In accordance with the present invention it is possible to grow phage in high-titre in a desired laboratory bacterium, but wherein phage are able to grow far less efficiently in a "wild-type" bacterium (i.e. not the desired laboratory bacterium). In this manner, the present invention provides a modified phage such as a modified lambda phage, which is capable of growing in a wild-type bacterium, such as E. coli, at least 1 x 10³ less efficiently than in a controlled laboratory setting. Desirably the phage is capable of growing at least 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸ or even 1 x 10⁹ less efficiently in a wild-type bacterium.

An exemplary modified phage is described in more detail in the examples section. As will be appreciated, the phage of the present invention are modified so as to be able to be efficiently grown in the laboratory under specifically controlled conditions, but if/when the phage are released into the wild, the phage are designed to not survive or survive to a lesser extent than un-modified phage or wild-type phage, as exemplified by the phage lambda wild-type (Hendrix and Duda, 1992). A preferred phage for use in growing industrially has a genome sequence as represented in Figure 15. The genome sequence shown in Figure 15 has been modified by deletion of certain sequences and introduction of appropriate Type-I and Type-ll restriction sites. The skilled reader will therefore appreciate that the sequence of the phage vector could in fact vary quite significantly from that shown in Figure 15. In this regard, Figure 14 refers to modifications which may be preferred and a preferred phage may comprise one or more, or all of the identified modifications.

The phage or phages of the present invention are typically further modified so as to be capable of expressing one or more antigenic or therapeutic peptides/proteins. The modifications may be such that the surface of the phage may also display by fusion or be conjugated to said peptide/protein and/or display by fusion or be conjugated to one or more peptides/proteins designed to target a receptor or receptors on the surface of cells. When administered to a subject, such as a human or other mammal, birds, or even fish and sharks, an immune response is elicited against said antigenic peptide/protein. A variety of uses for phage can be envisaged as described above and all which may be of relevance in terms of requiring large scale production of phage. Particularly suitable uses for phage are described in, for example, WO02/076498, WO2009130261 , WO2007015704, WO20081 15296 to which the skilled reader is directed. In accordance with the present invention, suitable phage may be modified so as to be reduced in their ability to replicate in the wild (i.e. outside the laboratory/production facility).

The phage to be administered to a subject may be designed to be immunogenic and/or therapeutic. Therapeutic phage may display (by coat protein fusion or conjugation) or express (from a eukaryotic expression cassette) one or more peptides or proteins designed to alleviate a disease or condition. Typically, this may be by way of a "gene therapy" type application well known in the art and described for example in WO9966061. Alternatively the phage may be designed to elicit an immune response in a subject. The immune response may be used to obtain antibodies to the particular peptide/protein for use in a separate application, such as the generation of antibodies from camels and sharks, which have special properties (Wesolowski et al., 2009; Conrath et a/., 2003; De Genst et al., 2006), or the immune response may be directly immunogenic, therapeutic or protective to the subject, against subsequent challenge by a disease causing agent as discussed, for example in WO02/07649. Thus, the compositions of the present invention may find application in the treatment and/or prevention of disease.

The phages of this invention may include any phage, phage derivative (such as cosmids or phages produced with the assistance of a helper phage/phagemid) or phage physically modified post production (e.g. by conjugating a foreign protein or peptide to the phage surface) produced for commercial purposes where the product is released into the environment and where it is desirable to restrict the replication of the phage or reduce the distribution of DNA from the phage product, once released into the "wild" i.e. outside the controlled phage production environment.

Detailed Description

The present invention will now be described by way of reference to the following non-limiting examples and figures which show: Figure 1 shows sequence remaining in an E. coli of the present invention following deletion of the mrcC to mrr gene sequences. Bases 4 574 986 to 4 585 836 have been removed;

Figure 2 shows sequence remaining in an E.coli of the present invention following fhuA deletion. Bases 167 534 to 169 431 have been removed;

Figure 3a (DLP12 prophage deletion) shows sequence remaining in an E.coli of the present invention following ybcN to ybcX deletion; Figure 3b shows sequence remaining after a second deletion in the DLP12 prophage (intD - ybcX) to remove remaining homology to bacteriophage NM1149. Bases 564 108 to 581 245 have been removed;

Figure 4a (RAC prophage deletion) shows sequence remaining in an E.coli of the present invention following kil and trkG deletion; Figure 4b shows sequence remaining after a second deletion in the RAC prophage (kil - ynaE) to remove remaining homology to bacteriophage NM1 149. Bases 1 416 101 to 1 432 210 have been removed;

Figure 5 (Qin prophage deletion) shows sequence remaining in an E.coli of the present invention following ydfK - ydfO deletion, removing all homology to bacteriophage NM1 149. Bases 1 630 494 to 1 635 421 have been removed;

Figure 6 shows sequence remaining in an E.coli of the present invention following fimB to fim deletion. Bases 4 539 032 to 4 547 683 have been removed;

Figure 7 shows sequence remaining in an E.coli of the present invention following ff/^'C to fli deletion. Bases 2 000 134 to 2 004 055 have been removed;

Figure 8 shows sequence remaining in an E.coli of the present invention following RecA deletion. Bases 2 820 780 to 2 821 741 have been removed;

Figure 9 lists the deletions involved in producing the host £ .coli strains described within;

Figure 10 shows an electron micrograph of a column-purified bacteriophage lambda preparation which is contaminated by fimbriae derived from the bacterial host; Figure 11 shows the result of a BLAST search for homology between the phage vector NM1 149 and the host E.coli BDEC-04, the strain produced after the second round of deletions from the host prophages;

Figure 12 shows a graphical representation of a Type-I restriction site;

Figure 13 shows sequence shows the general layout of sequence introduced at the multiple cloning site of a modified lambda phage of the present invention, containing additional type II sites for cloning and additional type I sites for safety;

Figure 14 lists the number of type I sites found in a modified lambda of the present invention, including both those that have already been characterised (14a) and those which can theoretically be deduced from the sequences of the known sites (14b). It is well established in the art (for example Fuller-Pace, ef a/., 1984; Nagaraja, et a/., 1985) that new type I restriction recognition sites can be created by recombination between the two recognition domains (Figure 12) of type I enzymes. The introduction of "hypothetical" type I restriction sites (i.e. those which are determined to be possible based on recombination between the two recognition domains found in the sequences avaialable) can also be incorporated into the genome of the phage vector to potentially increase the susceptibility of the phage to restriction by a wider range of wild-type bacterial strains;

Figure 15 shows the sequence of the entire genome of a modified phage according to the present invention; and

Figure 16 is Table listing non-essential genes which can be removed from lambda phage to improve safety, increase yields or increase cloning capacity of the vector.

All base numbers relating to the host E. coli are given are in accordance to the published sequence of G1655 (Gene Bank Accession No. U00096), which was used as a starting strain for all modifications. Example 1

The E. oli type I restriction system (EcoKI in E. coli K12 and its derivatives) is designed to prevent infection by phages by recognising specific sequences in the infecting phage DNA and digesting the phage DNA to stop phage replication. E. coli protects its own DNA by methylation i.e. the sequence recognised by the EcoKI enzyme is methylated by the hsdM subunit of the enzyme in the host DNA, which prevents restriction. Lambda DNA unmodified by the E. coli HsdM enzyme is thus susceptible to cleavage in an E. coli strain expressing the EcoKI enzyme.

The following experiments were performed to determine how much efficiency of plating is reduced when a phage with non-methylated DNA infects E. coli.

Example 1a

Strains used

Phage: Lambda NM1149-GFP - the phage lambda MN1149 (Murray, 1983) containing the expression cassette from the plasmid pEGFP-C1 (construction described in Clark and March, 2001). This phage contains 5 EcoKI sites.

Host strain: E. coli NM1056 (MG1655 A( ?sdS - mc/ . Both restriction and modification minus i.e. deleted for hsd and mcr.

Host strain: E. coli NM1049 MG1655 /ar.:cat. Both restriction and modification plus. Procedure

A 10mL culture of phage lambda NM1149-GFP was produced using E. coli NM1056 as a host, so the resulting phage were not modified (methylated). This was then used to infect both NM1056 and NM1049 E. coli strains, which were directly plated (using ten-fold serial dilutions) onto top agar plates as per standard procedures (Sambrook, Fritsch and Maniatis, 1989). A plaque (zone of clearing on the plate) is produced where a successful phage infection occurs. By comparing the number of plaques on restriction plus and restriction minus hosts, an estimation of the effect of restriction on the efficiency of plating is obtained. All experiments were performed in triplicate. Results

Number of plaques

NM1056 (R-M-) 2.3x10¹¹

3.3x10¹¹

2.8x10¹¹

Average 2.8x10¹¹

Number of plaques

NM1049 (R+M+) 7.7x10⁶

8.0x10⁶

9.6x10^s

Average 8.4x10⁶

This equates to a reduction of 3x10"

These results suggest that a phage which is produced in a non-methylated strain of E. coli will be 3x10⁴ times less able to infect a wild type host which is restriction positive for the relevant restriction enzyme.

Example 1 b

Strains used

Lambda NM1149-GFP. Phage contains 5 EcoKI sites.

E. coli BDEC-01. Strain produced in accordance with the present invention with custom deletions described in Figure 1 and Figure 2. Lacks fhuA, the T1 phage receptor and lacks the £coK system, the mcr system and the mrr system i.e. is restriction modification minus. This host differs from the one described in example 1a by additionally being deleted for the mrr restriction/modification system. E. coli G1655 fhu/K. The parent strain. Both restriction and modification plus and lacking fhuA, the T1 phage receptor.

Procedure

A 10mL culture of lambda NM1 149-GFP was produced using N 1056 as a host, so the resulting phage were not modified (methylated). This was then used to infect both MG1655 f u ^' and BDEC-01 , which were directly plated (using ten-fold serial dilutions) onto top agar plates as per standard procedures (Sambrook, Fritsch and Maniatis, 1989). A plaque (zone of clearing on the plate) is produced where a successful phage infection occurs. By comparing the number of plaques on restriction plus and restriction minus hosts, an estimation of the effect of methylation on efficiency of plating is obtained. All experiments were performed in triplicate.

Results

Number of plaques

.1 1

(R- -) 8.6x10

1.2x10 ,12

Average 9.8x10 |11

Number of plaques G1655 f/)uA^" (R+M+) 2.1x10⁶

2.2X10⁶

2.1 X10⁶

Average 2.1x10⁶

A reduction of 4.7x10⁵. These results suggest that a phage which is produced in a non-methylated strain of E. coli will be 4.7x10⁵ times less able to infect a wild type host.

Example 2: Generation of a mutant E. coli

Materials & methods

Starting strain

The strain used as the template for subsequent modifications is the sequenced MG1655 strain which was deposited with the Yale E. coli culture collection as CGSC7740.

Deletions which can be performed

The regions required to be deleted can be removed using patented Red/ET technology. A detailed description of the technology can be found in W099/29837 and WO01/04288 Four basic steps are involved: a) PCR-mediated generation of Amp/Cm/kn cassette flanked by homology arms. b) Red/ET rEcombination of the cassette into the loci mentioned below. c) Removal of the antibiotic selection marker by Cre or FLPe rEcombination. d) Verification of accurate performance of the Red/ET rEcombination mediated engineering step by PCR and sequencing.

This method does leave a scar on the E. coli genome (as shown in the following section), meaning some regions of internal homology will be introduced into the E. coli genome. All the deletions which have been carried out(and therefore introduced regions of homology) are so far apart as to make the risk of homologous recombination within the E. coli genome virtually zero. In the extremely unlikely event that recombination did occur, so much of the E. coli genome would be removed as to make the cells non-viable.

Stage 1 : a) RM system:4 574 986 to 4 585 836; mcrC - mrr

b) tonA (fhuA): 167 484 to 169 727; fhuA The 2 initial deletions mentioned in Stages 1 a and 1 b above were performed, to create MG1655 Stage 1 a and MG 1655 Stage 1 b, as described above and with reference to the sequence information shown hereinafter. These remove this type I restriction modification system and the fhuf gene which codes for the outer membrane protein which is the T1 phage receptor. The MG1655 Stage 1 a and Stage 1 b strains are therefore resistant to infection by T1 , a common and highly virulent contaminant of E. coli cultures.

Stage 2: a) DLP 12 571 760 to 581 245; ybcN - ybc

b) Rac 1 416 101 to 1 423 191 ; kil - trkG

c) Qin 1 630 494 to 1 635 421 ; yoYK - ydfO

The further 3 deletions mentioned in Stages 2a, 2b and 2c above can be performed, to create MG 1655 Stage 2a, Stage 2b and Stage 2c, as described above and with reference to the sequence information shown hereinafter. These will remove all or some regions of the 3 lambdoid prophages in the G1655 genome which have homology to phage lambda.

Stage 3: a) DLP 12 Extends deletion 2a from 564 108 to 581 245; intD - ybcX

b) Rac Extends deletion 2b from 1 416 101 to 1 432 210; kil - ynaE

The further 2 deletions mentioned in Stages 3a and 3b above can be performed, to create MG 1655 Stage 3a and Stage 3b, as described above and with reference to the sequence information shown hereinafter. These will remove all or some regions of the 3 lambdoid prophages in the G1655 genome which have homology to phage lambda. These deletions remove the remaining significant homology to many lambda cloning vectors (for example all significant homology (homology of more than 20 base pairs) to lambda NM 1149 is removed).

Stage 4: Fimbiae 4 539 032 to 4 547 683; f/mB to firri

This deletion removes the genes present in the host E. coli which are responsible for the production of fimbriae. Removal of fimbriae will simplify the purification of progeny phage from the milieu of lysed bacteria by greatly reducing the amount of contaminating protein present. Fimbriae are proteinaceous structures of a similar size to bacteriophages and could co-purify under certain conditions. This is illustrated in Figure 10, showing an electron micrograph of column purified bacteriophage preparation, which is contaminated by fimbriae from the host bacteria. Additionally, when they are present on the cell surface, fimbriae promote the aggregation of bacteria and bacterial debris, which could hamper purification.

Stage 5: Flagella 2 000 134 to 2 004 055; fliC to fill

This deletion removes the gene for the major flagellar subunit, without which flagella will not be produced. Removing the flagella reduces the amount of contaminating protein present, simplifying purification. Additionally, flagellar proteins are highly immunostimulatory and removing them from the host reduces the risk of a small amount of contamination by proteins which could affect the immune responses observed.

Stage 6 : f?ecA 2 820 730 to 2 821 791 ; RecA

After completion of the first 2 stages of deletions, an optional further deletion can be carried out to remove the recA gene - this will be performed to create MG1655 Stage 5 strain. This will greatly reduce homologous recombination, which could have use in certain applications, such as improving efficiency of gene cloning or preparing seed stocks of phage constructs.

Example 3: Generation of modified lambda phage

The purpose of designing a modified lambda phage is to provide a phage which is able to be grown efficiently under controlled laboratory/industrial conditions, but which is inefficient at growth in a wild-type environment.

The phage described herein (termed BD03) is based on a modified cloning vector lambda NM1 149 (Murray, 1983).

Lambda BD03 has the following characteristics:

The number of type II restriction sites has been increased to allow easier manipulation (see Figure 13 for details). The number of type I sites (both known and currently hypothetical) has been increased (Figure 13 and 14). The number of sites for the known EcoAI recognition sequence has been increased to at least 6 sites per genome.

It has been deleted for the restriction alleviation protein (ral) further increasing the sensitivity of the phage to restriction by the bacterial type I system.

It is deleted for the entire lysogeny region. All the genes from orf314 to ea8.5 are removed from the BD03 vector. Lambda phage lacking the lysogeny region will be considerably less viable than wild-type lambda.

It does not express the tail fibre genes. BD03 has had the tail fibre genes, found in wild-type lambda phage deleted. The tail fibre increases the number of receptors that lambda pahge can bind to on the E. coli cell surface and greatly increases the binding efficiency of the phage for the host. In the wild this provides a significant advantage. The reduced ability of lambda phage to infect E. coli is demonstrated in Hendrix and Duda, 1992. The advantages of retaining the tail fibre genes in allowing the phage to grow more efficiently in diverse environments is discussed in Gallet ef a/., 2009.

Figure 15 provides the complete sequence of the phage vector BD03.

Example 4: increased susceptibility of a modified lambda phage to type I digestion

Previous examples have detailed the susceptibility of lambda phage (which have been prepared on a restriction/modification deficient host phage) to type I digestion by a wild-type host. The lambda vector BD03 has had several type I restriction sites (Figure 13 and Figure 14a) added to increase its susceptibility to digestion. To confirm a reduced viability, phage without (BD02) and with (BD03) added restriction sites as described in Figure 13 were grown on NM65, an E. coli host which expresses the EcoA type I restriction system. BD02 contains 1 EcoA site and BD03 contains 6 EcoA sites.

Seed stocks of lambda BD02 and lambda BD03 were obtained. Both were grown in BDEC-02 (as described in Figure 9) an r-m- host, producing progeny phage that were not methylated. Both phage were then subsequently titred on BDEC-02 and E. coli NM65 (EcoA+). Results averaged over 3 repeat runs

These results demonstrate the increased sensitivity of a phage containing artificially added type I restriction sites, grown on a methylation deficient host, to digestion by a wild-type bacterial host.

References cited

Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley , Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. 1997 The complete genome sequence of Escherichia coli K-12. Science. 277: 1453-62.

Clark JR, March JB. 2001. Bacteriophage-mediated nucleic acid immunization. FEMS Imm. Med. Micro. 1652: 1-6.

Conrath KE, Wernery U, Muyldermans S, Nguyen VK. 2003 Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol. 27: 87-103. Review.

De Genst E, Saerens D, Muyldermans S, Conrath K. 2006 Antibody repertoire development in camelids. Dev Comp Immunol. 30: 187-98.

Fuller-Pace FV, Bullas LR Delius H, Murray, NE. 1984. Genetic recombination can generate altered restriction specificity. PNAS. 81 : 6095-99.

Gallet R, Shao Y, Wang IN. 2009. High adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment. BMC Evol. Biol. 9: 241-253.

Hendrix RW, Duda RL. (1992). Bacteriophage lambda PaPa: not the mother of all lambda phages. Science. 258: 1145-8.

Mizoguchi H Tanaka-Masuda K, Mori H. 2007 A simple method for multiple modification of the Escherichia coli K-12 chromosome. Biosci Biotechnol Biochem. 71 : 2905-1 1.

Murray NE. 1983 in Lambda II (Hendrix, R.W., Roberts, J.W., Stahl, F.W. and Weisberg, R.A., eds ), pp. 395-431, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

Murray NE. 2000. Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle). Microbiol Mol. Biol. Rev. 64: 412-34.

Nagaraja V, Shepherd JC, Bickle TA. 1985. A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity. Nature. 316: 371-2.

Pc-sfai G, Plunkett G 3rd, Feher T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, Blattner FR. 2006 Emergent properties of reduced-genome Escherichia coli. Science. 312: 1044-6.

Sambrook, Fritsch and Maniatis. 1989 Molecular Cloning A Laboratory manual: Third Edition Cold Spring Harbour Laboratory Press.

Titheradge AJB, King J, Ryu J, Murray NE. 2001. Families of restriction enzymes: an analysis presented by molecular and genetic data for type ID restriction and modification systems. Nucleic Acids Res. 29: 4195-205.

Wang K, Liu CQ, Cao H, Chen XF. 2006 Insight into relationship among mRNA coding region length, folding tendency and codon usage bias in Escherichia coli. Wei Sheng Wu Xue Bao. 46: 895-9. Wesolowski J, Alzogaray V, Reyelt J, Unger , Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. 2009 Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 198: 157-74.

Claims

What is claimed:

1. A method of obtaining bacteriophage for commercial purposes, the method comprising:

(a) growing a bacterium which is deficient in one or more Type I restriction and/or modification genes; and/or deficient for one or more genes ecoding for fimbriae and/or flagella protein(s); and

(b) infecting said bacterium with a suitable phage in order to allow multiplication of said phage; or inducing growth of a prophage which has previously been integrated into the genome of the bacterium, in order to allow multiplication of phage .

2. The method according to claim 1 , further comprising the step of isolating the phage and formulating into a product.

3. The method according to claim 2 wherein the product is to be administered to a subject for therapeutic or prophylactic purposes, or is to be applied in the environment.

4. The method according to any preceding claim wherein the bacterium is an E.coli, Salmonella species, such as Salmonella Typhimurium, Shigella species, such as Shigella Flexneri, Enterococcus species, such as Klebsiella, Proteobacteria species, such as Pseudomonas species. Haemophilus species, and Firmicutes species, such as Bacillus species and Streptococcus species.

5. The method according to any preceding claim wherein the bacterium is further modified so as to delete one or more prophage sequences from the genome of the bacterium.

6. The method according to any preceding claim wherein less than 4% of a parent bacterium's genome has been deleted in order to generate the bacterium for use in obtaining the bacteriophage.

7. The method according to any preceding claim wherein the bacterium is an E.coli dervied strain.

8. The method according to claim 7, wherein the E.coli is derived from strain MG1655.

9. The method according to either of claims 7 or 8 wherein said E.coli comprises one or more of the following genes/nucleic acid sequences which have been rendered deficient:

fluA; one or more genes associated with fimbriae and for flagella production, such as flagellin structural proteins, yg/L (a putative fimbrial like protein, flgA-H (genes associated with flagellin biosynthesis); flhA-E (genes associated with flagellin synthesis chemotaxis; fli A-R (genes associated with flagellin biosynthesis); fimB- (genes associated with fimbriae biosynthesis); one or more endonuclease genes, such as end. A; one or more DNA repair genes, such as rec A; one or more toxic genes/proteins, such as hok A, hok C, hok E and mok C; and/or one or more genes associated with auto-aggregation, such as flu (antigen 43).

10. The method according to any preceding claim wherein the modified bacterium comprises a modified phage binding target, such that wild-type phages cannot infect the host cell.

11. The method according to any preceding claim, wherein the bacterium has been modified to lack nucleic acid sequence(s) which is/are homologous with the bacteriophage.

12. The method according to any preceding claim wherein the bacteriophage or prophage has been modified by the introduction of one or more Type I restriction sites.

13. The method according to claim 12 wherein the bacteriophage/prophage is an E.coli bacteriophage, which has been modified to include one or more of the following Type-I recognition sequences: £co377l, Eco394l, Eco585l, £co646l, Eco777l, £co826l, Eco851 l, £co912l, EcoAI, £coBI, EcoDI, EcoDXXI, EcoEI, EcoKI, EcoPrrl, EcoR124l, EcoR124ll, SfyLTIII, SrySBLI, SfySEAI, SrySENI, SrySGI, SfySJI, SfySKI, SrySPI and SfySQI.

14. The method according to claims 12 or 13, wherein the bacteriophage/prophage is a lamboid phage.

15. The method according to any one of claims 12-14 wherein the bacteriophage/prophage has been modified to lack one or more restriction modification affecting proteins.

16. The method according to any one of claims 12-15 wherein the bacteriophage/prophage has been modified in order to render deficient one or more genes responsible for lysogeny and/or bacteriophage tail-fibres.

17. The method according to claim 16 wherein the lamboid phage genome has been modified to lack or render deficient one or more of the sequences identified in Figure 16.

18. The method according to claim 17 wherein the lamboid phage genome has been modified to lack or render deficient 2, 3, 4, 5, 6, 7, 8, 9, 10 or all identified sequences in Figure 16.

19. The method according to any of claims 13-18 wherein the bacteriophage/prophage has been modified to include one or more Type II restriction sites in addition to any which may have been present prior to modification.

20. The method according to any of claims 13-19 wherein the bacteriophage/prophage has been modified to include one or more artificial Type-I restriction sites.

21 . The method according to any preceding claim wherein the bacteriophage/prophage has been modified by altering its nucleic acid sequence to minimise homologous recombination with the host bacterium's genome (less than 25 nucleotides?)

22. The method according to any preceding claim wherein the bacteriophage/prophage has been modified so as to express one or more exogenous nucleic acid sequences, which encode an antigenic or therapeutic nucleic acid(s)/peptide(s)/protein(s).

23. Use of a bacterium which is deficient in one or more restriction and/or modification genes for growing phage.

24. A modified bacterium which is deficient in one or more restriction and/or modification genes; and/or deficient for one or more genes ecoding for fimbriae and/or flagella protein(s); and wherein one or more, or all prophage sequences have been removed from the bacterium's genome and wherein the bacterium comprises less than 4% of a reduction in size (in terms of bases removed) compared with the parent strain from which it is derived.

25. The modified bacterium according to claim 24 which is a modified E.coli.

26. The modified E.coli according to claim 25 were the said E.coli comprises one or more of the following genes/nucleic acid sequences which are rendered deficient:

/7uA; one or more genes associated with fimbriae and/or flagella production, such as flagellin structural proteins, ygIL (a putative fimbrial like protein, 7gA-H (genes associated with flagellin biosynthesis); //ΛΑ-Ε (genes associated with flagellin synthesis/chemotaxis; f/mB-H (genes associated with fimbriae biosynthesis); fli A-R (genes associated with flagellin biosynthesis); one or more endonuclease genes, such as end. A; one or more DNA repair genes, such as rec A; one or more toxic genes/proteins, such as hok A, hok C, hok E and mok C; and/or one or more genes associated with auto-aggregation, such as flu (antigen 43)

27. The modified bacterium according to any of claims 24 -26 wherein the modified bacterium comprises a modified phage binding target

28. The modified bacterium according to any of claims 24 - 27 which has been modified to lack nucleic acid sequence(s) which is/are homologous with the bacteriophage

29. A bacteriophage which has been modified so as to be capable of growing in a wild-type host bacterium at least 1 x 10³ less efficiently than in a modified bacterium according to any one of claims 24 - 28

30. The bacteriophage accoding to claim 29 which has been modified by the introduction of one or more Type I restriction sites.

31. The bacteriophage according to claim 30 wherein the bacteriophage/prophage is an E.coli bacteriophage, which has been modified to include one or more of the following Type-I recognition sequences: Eco377l, £co394l, Eco585l, Eco646l, Eco777l, Eco826l, Eco851 l, £co912l, EcoAI, EcoBI, EcoDI, EcoDXXI, EcoEI, EcoKI, EcoPrrl, EcoR124l, EcoR124ll, SryLTHI, SfySBLI, SrySEAI, SfySENI, SfySGI, SfySJI, SrySKI, SrySPI and SfySQI.

32. The bacteriophage according to any of claims 29 - 31 , wherein the bacteriophage is derived from a lamboid phage.

33. The bacteriophage according to any one of claims 29 - 32 wherein the bacteriophage has been modified to lack one or more restriction modification affecting proteins.

34. The bacteriophage according to any one of claims 29 - 33 wherein the bacteriophage has been modified in order to render deficient one or more genes responsible for lysogeny and/or bacteriophage tail-fibres.

35. The bacteriophage according to claim 34 wherein the lamboid phage genome has been modified to lack or render deficient one or more of the sequences identified in Figure 16.

36. The bacteriophage according to claim 35 wherein the lamboid phage genome has been modified to lack or render deficient 2, 3, 4, 5, 6, 7, 8, 9, 10 or all identified sequences in Figure 16.

37. The bacteriophage according to any of claims 29 - 36 wherein the bacteriophage has been modified to include one or more Type II restriction sites in addition to any which may have been present prior to modification.

38. The bacteriophage according to any of claims 29 - 37 wherein the bacteriophage has been modified to include one or more artificial Type-I restriction sites.

39. The bacteriophage according to any of claims 28 - 38 claim wherein the bacteriophage has been modified by altering its nucleic acid sequence to minimise regions of homology with the host bacterium's genome.

40. The bacteriophage which is at least 90%, 95%, 97%, or 99% identical to the sequence shown in Figure 15.

41. The bacteriophage according to any of claims 29 - 40 wherein the bacteriophage has been modified so as to express one or more exogenous nucleic acid sequences, which encode an antigenic or therapeutic nucleic acid(s)/peptide(s)/protein(s).