WO1991017256A1 - Souches resistantes a l'autolyse et procedes faisant appel a ces souches - Google Patents

Souches resistantes a l'autolyse et procedes faisant appel a ces souches Download PDF

Info

Publication number
WO1991017256A1
WO1991017256A1 PCT/US1991/003258 US9103258W WO9117256A1 WO 1991017256 A1 WO1991017256 A1 WO 1991017256A1 US 9103258 W US9103258 W US 9103258W WO 9117256 A1 WO9117256 A1 WO 9117256A1
Authority
WO
WIPO (PCT)
Prior art keywords
lyt
plasmid
gene
strains
strain
Prior art date
Application number
PCT/US1991/003258
Other languages
English (en)
Inventor
Alexander Tomasz
Original Assignee
The Rockefeller University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Rockefeller University filed Critical The Rockefeller University
Publication of WO1991017256A1 publication Critical patent/WO1991017256A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • This invention relates to autolysis resistant
  • this invention relates to pneumococcal strains having the gene which produces autolysin interrupted by an inserted plasmid so that the enzyme is not produced and organisms comprising said strains do not self-destruct; and, this invention relates to processes for making and employing such strains. More generally, this invention relates to a method for preventing the expression of undesired gene products during the growth of a cellular microorganism without substantially affecting the other aspects of its phenotype which comprises insertional inactivation of the appropriate gene(s) in the microorganism, and growing the microorganism in an appropriate medium.
  • this invention relates to improvements in a method for obtaining desired gene products from a growing cell by inhibiting the expression of undesired gene products through insertional inactivation of the appropriate gene(s) in the cell and growing the cell in an appropriate medium.
  • DNA cloning and methods for constructing recombinant DNA techniques are known, see, e.g., Goodenough, U.,
  • a plasmid or phage cloning vehicle or vector which can be cut by a restriction enzyme such as EcoRl without losing its ability to self-replicate.
  • cloning vehicles include Col El, pSClOl.
  • the cloning vehicle is linearized by the restriction enzyme; and, this "linearized plasmid" is next treated with an exonuclease which attacks and digests the 5' ends of duplex DNA to create single- stranded "tails" at the 3' ends.
  • exonucleases include those derived from phage ⁇ . Tracts of poly-adenine (poly-A) are then added to these tails.
  • This addition reaction can be mediated by an enzyme terminal transferase.
  • the DNA to be cloned i.e., the plasmid or recombinant plasmid is similarly treated with exonuclease and terminal transferase: the terminal transferases add tracts of poly-thymine (poly-T) to the 3' ends of the recombinant plasmid.
  • the preparations of the cloning vehicle and the recombinant plasmid are admixed: the poly-A tracts anneal with the poly-T tracts to form a circular structures; and, in a final step, are covalently sealed with ligase.
  • Pneumococci have remained major human pathogens in spite of the introduction of antibacterial agents such as penicillin into chemotherapy. There is general agreement that while the rate of mortality from
  • pneumococcal disease has declined, the attack rate has remained unchanged and morbidity and even mortality form this bacterial disease plateaued off at unacceptably high levels (e.g., 30% for meningitis), in spite of the availability and general use of antibiotics.
  • the importance of finding more effective interventions against pneumococcal disease has gained emphasis by the emergence and worldwide spread of penicillin-resistant and multi-resistant penumococcal strains and by the shift in the patient populations (in the Western world) in the direction of a higher proportion of the aged who are known to be particularly susceptible to pneumococcal disease.
  • intervention of pneumococcal infection through vaccination has remained an important, even if so far unfulfilled, public health goal all over the world.
  • Streptococus pneumoniae is used in the production of antigenic polysaccharides to prepare vaccines against S. pneumoniae infection.
  • microorganisms which express or which are caused to express e.g., by
  • maximal cell density may be achieved by growing the microorganisms into the
  • pathogenic strains of S. pneumoniae belong to any one of a large number (83) of different capsular types, most of which act as distinct antigens in a human host.
  • antibodies produced against a strain expressing capsular polysaccharide type 3 will not react with another strain that expresses capsular polysaccharide type 2 on its surface.
  • all effective antipneumococcal vaccines are multi-component vaccines, composed of mixtures of chemically distinct capsular polysaccharides.
  • one of the vaccines includes polysaccharides from 23 different strains which represent over 95% of pathogenic pneumoccocci. This vaccine is marketed as Pneumovax R 23 (Merck).
  • the producer cultures are allowed to grow to maximal cell density, e.g., into the stationary phase of growth, in order to have optimal yield of material.
  • maximal cell density e.g., into the stationary phase of growth
  • polysaccharides is usually conducted to make them available for the production of vaccines.
  • the first step is the separation of the producing bacteria from the culture medium since the latter contains most of the capsular polysaccharides in soluble form.
  • the broth is then further fractionated for the isolation of the bacterial polysaccharides.
  • microorganisms carry a gene for the production of autolysin.
  • this gene is the lytA gene, which, when expressed, causes the microorganism to produce the enzyme called autolysin.
  • the autolysin becomes activated causing destruction of the cell wall, i.e., the organism lyses and dies.
  • Autolysis releases toxic (inflammatory) materials (e.g., cell wall materials) into the culture medium thereby contaminating
  • polysaccharide vaccine preparations The products of autolysis are inflammatory and can cause adverse reactions such as erythema and induration at injection site, fever, rashes, hives, vomiting, diarrhea, and even, in some cases, anaphylactoid reactions.
  • polysaccharide preparations are contaminated by cell wall material which is released from the bacteria due to the triggering of the activity of the major pneumococcal autolytic enzyme (the N- acetylmuramoyl-L-alanine amidase, to be referred to as "amidase") in the stationary phase of growth.
  • the major pneumococcal autolytic enzyme the N- acetylmuramoyl-L-alanine amidase, to be referred to as "amidase”
  • pneumococci is highly inflammatory as shown in several models of pneumococcal disease.
  • polysaccharide vaccines at least 51% of the individually tested experienced adverse reactions. These adverse reactions are a result of contaminants in the vaccines, particularly cell wall material in the vaccines.
  • the origin of the contaminating wall material in the vaccine preparations is the process of autolysis: the major agent of which is a cell wall degrading enzyme, a so-called autolysin. As more fully discussed herein, this enzyme is ubiquitous in all natural strains of pneumococci and its destructive activity may be
  • the culture medium contains soluble cell wall material released by the triggering of autolysin.
  • polysaccharides and of the cell wall materials are sufficiently similar such that removal of the latter through purification techniques is difficult, costly, and not sufficiently effective.
  • the lytA gene has been isolated, and a plasmid has been inserted into S. pneumoniae which has resistance to erythromycin as a marker and which interrupts the gene for producing lysin.
  • a plasmid has been inserted into S. pneumoniae which has resistance to erythromycin as a marker and which interrupts the gene for producing lysin.
  • Tomasz, A. et al "Insertional Inactivation of the Major Autolysin Gene of Streptococcus pneumoniae," 170 J. Bacteriology 5931-5934 (Dec. 1988), incorporated herein by reference.
  • Tomasz et al do not teach or suggest any methods of employing this plasmid, for example, for improving the production of useful products from
  • microorganisms by extending the useful life of said microorganisms by preventing them from autolysing, prematurely dying, and contaminating the broth.
  • the genetically altered strains (with inactivated lytA) must include substantially all relevant producer
  • strains i.e., the method of rendering the lytA gene inactive must be applicable to substantially all
  • pneumonococcal strains with the genetically inactivated lytA marker must be fully viable and able to grow at substantially the same rate and in the same media used with lytA
  • inactivated LytA strains must be capable of producing vaccines which provide little or no adverse reactions (especially in comparison to the 51-71% rate of adverse reactions experienced from presently available
  • This invention provides strains, especially
  • pneumococcal strains having a plasmid inserted therein which results in the gene producing lysin or autolysin, especially the lytA gene, being interrupted so that the enzyme is not produced and organisms comprising said strains do not self-destruct.
  • This invention also provides processes for making and employing such
  • this invention provides a method for preventing the expression of undesired gene products during the growth of a cellular microorganism without substantially effecting the phenotype which comprises inserting an altered plasmid in the
  • this invention provides an improvement in a method for obtaining desired gene products from a growing cell especially
  • autolysin also known as the autolytic enzyme N-acetylmuramoyl-L-alanine amidase
  • the plasmid preferably interrupts the lytA coding sequence, and more preferably carries the marker of resistance to erythromycin.
  • Fig. 1 illustrates the growth rate of pneumonococcal with insertionally inactivated autolysin.
  • Fig. 2 depicts daughter cell separation at the end of cell division in pneumococcal Lyt- with insertionally inactivated autolysin.
  • Insertional inactivation of the lytA gene is brought about by interrupting the lytA coding sequence by the insertion of a plasmid or a gene, particularly ermC, a staphylococcal gene conferring resistance to erythromycin; and, this resistance to erythromycin is useful as a marker.
  • the plasmid comprises an altered plasmid having a detectable marker.
  • the desired products comprise pneumonococcal
  • the undesired gene products comprise autolysin or lysin
  • the altered plasmid comprises an interrupted lytA coding sequence
  • the marker is resistance to erythromycin.
  • EXAMPLE 1 Determination of presence and expression of lytA gene in all relevant producer strains of S. pneumoniae. The structural determinant of autolysin or the
  • amidase i.e., the lytA gene
  • E. coli E. coli
  • DNA representing the lytA gene cloned in the vector plasmid was radiolabeled ( 32 p) using routine procedures for the preparation of DNA probe.
  • This probe was then used to determine whether or not the strains set forth in Tables A, B and C below carry the lytA gene.
  • the strains of Tables A, B and C were cultured on blood agar plates to form separate colonies.
  • each colony was then grown to a liquid culture, lysed and the DNA transferred to membrane filters and probed with the 32 p-labeled lytA DNA probe and autoradiography. If the lytA gene was present in the DNA of a strain, the radiolabeled probe would be hybridized (annealed) to the complementary DNA on the membrane filters, resulting in a positive signal upon autoradiography. All of the pneumococcal strains tested positively for the lytA gene. The strains in
  • Tables A, B and C include the relevant vaccine producer strains.
  • Plasmid pGL80 carrying the lytA gene (See Garcia et al, FEMS Microbiola Lett. 29:77-81 (1985), incorporated herein by reference) was cut with Tapl and ligated to the 2.0-kilobase Mspl fragment of pE194 containing the ermC gene (See Horinouchi et al, J. Bacteriol.,
  • the rate of adjustment to steady-state doubling times was tested in the following situations: a shift from 30 to 37 C and vice versa; a shift from poor to rich medium; and a shift from the stationary to the
  • Fig. 1 depicts the results of this testing by showing the growth rate of the pneumococcal mutant with insertionally inactivated autolysin.
  • Fig. 1 cultures of the pneumococcal Lyt- mutant RUP24 (lyta:ermC) and its isogenic Lyt + parent R6x were grown in the aforementioned chemically defined medium (A) or in casein hydrolysate medium suplemented with yeast extract (See Lacks et al, Biochim. Biophys. Acta 39:508-17 (1980), incorporated herein by reference) (B) at 37°C. Cultures of the Lyt- deletion mutant M31 (lytA) were also grown under the same conditions. In the experiments illustrated in panel B, growth
  • Lyt- inseritonal mutants plated on the surface of blood agar produced normal alpha-hemolytic zones around the colonies indistinguishable from those surrounding colonies of Lyt + cells.
  • Isogenic (Lyt + - Lyt-) strains were constructed from encapsulated pneumococci in the following manner:
  • the Lyt- (Em r ) marker from RUP1 was introduced by genetic transformation into a type 6 clinical isolate (strain A112).
  • the capacity to produce type 3 capsule was transformed into the isogenic pair of R6x (Lyt + )-RUP24 (Lyt- Em r ) cells by using transforming DNA isolated from strain HB 565 as the donor of type 3 capsular
  • mice consecutive steps in mice in the following manner.
  • mice were challenged with large inocula (10 7 to 10 8 ) of a given strain and killed 24 hour later.
  • the spleens were aseptically removed, homogenized, suspended in tryptic soy broth supplemented with glucose and yeast extract,and incubated at 37°C for 16 to 18 hour. Such a culture was then used to inoculate a second set of animals.
  • groups of 10 mice were challenged with a series of inocula from each strain and survival rates of mice were monitored by daily observation. The results of this challenge are set forth in Table 3.
  • Isogenic (Lyt + - Lyt-) strains are constructed as set forth in Example 2 (vi). Cultures of RUP20 cps6A Lyt-, RUP 21 cps 6A Lyt + , RUP25 cps3 Lyt " and RUP26 cps3 Lyt + are grown in a liquid nutrient medium or culture broth. Maximal cell density is achieved in all cultures and at the end of the exponential growth phase, the producer strains are removed from the culture media. The
  • resulting culture supernatants contain, inter alia, the capsular polysaccharides.
  • the capsular polysaccharides are isolated from the culture supernatants by membrane filtration and precipation with
  • polysaccharides are then dissolved in a lactose
  • Lyt + - Lyt- vaccines are administered subcutaneously: 1 ml (0.15 ug
  • a second group is given cps6A (Lyt + ) vaccine; a third group is given cps3 (Lyt-) vaccine; and, a fourth group is given cps3 (Lyt + ) vaccine.
  • mice Three weeks after vaccination, these mice are challenged with series of inocula from strains they are vaccinated against, in accordance with the procedure of Example 2 (vi) such that of each group of thirty mice ten are given inoculum of size 10 4 CFU, ten are given inoculum of size 10 5 CFU, and ten are given inoculum of size 10 6 CFU, and, in each group of ten, 5 mice are given the Lyt- strain and 5 are given the Lyt + strain, e.g., ten of thirty mice given cps6A (Lyt-) vaccine are challenged with inoculum of size 10 4 CFU, five of which are challenged with RUP20 cps6A Lyt- and five of which are challenged with RUP21 cps6A Lyt + . All of the mice survived; antibodies are produced from the administration of the vaccines.
  • mice given vaccines from Lyt- cultures experience at least one of the following adverse
  • polysaccharide(s) per ml with a typical injection having a volume of 0.5 ml.
  • Doses to be administered can be accordingly adjusted by the skilled artisan taking into account such factors as the age, weight, and condition of the patient being vaccinated.
  • Antibodies induced by polysaccharide vaccines may persist for as long as 42 to 48 months or longer after vaccination.
  • Autolysis increases the production steps required to make such products by causing the need for purification; autolysis thereby increases the cost of such products.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Sont décrits et revendiqués une méthode pour empêcher l'expression de produits du gène non désirés et un procédé visant à améliorer la méthode qui permet d'obtenir les produits désirés à partir de l'expression de microorganismes cellulaires en croissance, qui font appel tous deux à l'insertion d'un plasmide modifié. Sont décrits en particulier: une méthode pour empêcher l'expression de l'enzyme autolytique (l'amidase de N-acétylmuramoyl-L-alanine) de Streptococcus pneumoniae; une méthode pour introduire le gène d'autolysine inactivé dans l'une quelconque des couches de Streptococcus pneumoniae avec des capsules chimiquement différentes; un procédé permettant d'utiliser ces pneumococci non autolysants pour produire des polysaccharides capsulaires non contaminés par des matériaux de parois cellulaires, ce qui permet d'obtenir un vaccin antipneumocoque plus sûr et meilleur marché.
PCT/US1991/003258 1990-05-09 1991-05-05 Souches resistantes a l'autolyse et procedes faisant appel a ces souches WO1991017256A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US52128490A 1990-05-09 1990-05-09
US521,284 1990-05-09

Publications (1)

Publication Number Publication Date
WO1991017256A1 true WO1991017256A1 (fr) 1991-11-14

Family

ID=24076130

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/003258 WO1991017256A1 (fr) 1990-05-09 1991-05-05 Souches resistantes a l'autolyse et procedes faisant appel a ces souches

Country Status (1)

Country Link
WO (1) WO1991017256A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834233A (en) * 1992-04-06 1998-11-10 Gx Biosystems A/S Method of limiting the survival of genetically engineered microorganisms in their enivronment
WO1999057281A2 (fr) * 1998-05-06 1999-11-11 St. Jude Children's Research Hospital Antibiotiques peptidiques inhibant la croissance de pneumocoques, proteines de streptocuccus pneumoniae transporteurs d'abc et comprenant un systeme de transduction du signal a deux elements, et procedes d'utilisation desdits antibiotiques
US6331407B1 (en) 1998-05-06 2001-12-18 St. Jude Children's Research Hospital Antibiotics and methods of using the same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY, Vol. 57, No. 7, issued July 1989, BERRY et al., "Reduced Virulence of a Defined Preumolysin-Negative Mutant of Streptococcus Pneumoniae", pages 2037-2042. *
JOURNAL OF BACTEROLOGY, Vol. 170, No. 12, issued December 1988, TROMASZ et al., "Insertional Inactivation of the Major Autolysin Gene of Streptococcus Pneumoniae", pages 5931-5934. *
MOL. GEN. GENET., Vol. 204, issued 1986, LOPEZ et al., "Isolation, Characterization and Physiological Properties of an Antolytic-Defined Mutant of Streptococcus Pneumoniae", pages 237-242. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834233A (en) * 1992-04-06 1998-11-10 Gx Biosystems A/S Method of limiting the survival of genetically engineered microorganisms in their enivronment
US6017730A (en) * 1992-04-06 2000-01-25 Gx Biosystems A/S Method of limiting the survival of genetically engineered microorganisms in their environment
WO1999057281A2 (fr) * 1998-05-06 1999-11-11 St. Jude Children's Research Hospital Antibiotiques peptidiques inhibant la croissance de pneumocoques, proteines de streptocuccus pneumoniae transporteurs d'abc et comprenant un systeme de transduction du signal a deux elements, et procedes d'utilisation desdits antibiotiques
WO1999057281A3 (fr) * 1998-05-06 2000-03-30 St Jude Childrens Res Hospital Antibiotiques peptidiques inhibant la croissance de pneumocoques, proteines de streptocuccus pneumoniae transporteurs d'abc et comprenant un systeme de transduction du signal a deux elements, et procedes d'utilisation desdits antibiotiques
US6331407B1 (en) 1998-05-06 2001-12-18 St. Jude Children's Research Hospital Antibiotics and methods of using the same
US6448224B1 (en) 1998-05-06 2002-09-10 St. Jude Children's Research Hospital Antibiotics and methods of using the same

Similar Documents

Publication Publication Date Title
Tomasz et al. Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae
Hodgson Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes
CA1259934A (fr) Vaccins vivants non revertants contre des salmonella
Clewell et al. Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918)
EP0381706B1 (fr) Procede permettant de maintenir un gene recombinant desire dans une population cellulaire genetique
US4337314A (en) Genetically attenuated bacterial vaccines with multiple mutations of the same phenotype
Ike et al. Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice
US4550081A (en) Non-reverting salmonella
HU205262B (en) Process for producing new, nonreverting living vaccine and stock against shigella
US4681762A (en) Genetically attenuated bacterial vaccines with multiple mutations of the same phenotype
US5840312A (en) Recombinant Bacillus anthracis strains unable to produce the lethal factor protein or edema factor protein
Muckerman et al. Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae
Mallavia et al. The genetics of Coxiella burnetii: etiologic agent of Q fever and chronic endocarditis
US7597896B2 (en) Composition comprising a protein encoded by a modified ERP gene of Mycobacterium
JP4495143B2 (ja) 生物学的安全性が向上した経口ワクチン接種用凍結乾燥形態のコレラ菌弱毒化株
WO1991017256A1 (fr) Souches resistantes a l'autolyse et procedes faisant appel a ces souches
Clarke et al. Galactose epimeraseless mutants of Salmonella typhimurium as live vaccines for calves.
Bandara et al. Putative outer membrane autotransporter protein influences survival of Brucella suis in BALB/c mice
AU739191B2 (en) Vaccines containing attenuated bacteria
US6905691B1 (en) Vaccines containing attenuated bacteria
AU2270799A (en) Tgc method for inducting targeted somatic transgenesis
Gemski et al. Isolation and characterization of minicell-producing mutants of Shigella spp
KR20230159560A (ko) 장독소원성 박테로이데스 프라길리스의 증식을 억제하기 위한 조성물 및 방법
Le Roux12 et al. Construction of a Vibrio splendidus Vsm metalloprotease mutant using a novel counter-selectable suicide vector
Stone An examination for phage-mediated genetic transfer mechanisms in Staphylococcus aureus.

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA