Recombinant listeria

Details Recombinant listeria Recombinant listeria

2000-06-06

2004-11-30

Paras, Jr., Peter (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

Reexamination Certificate

active

06825028

ABSTRACT:

The object of the invention is a method for inducting targeted somatic transgenesis (TGC=targeted genetic conditioning), which is used for expressing foreign proteins in cells, tissue, organ or an entire host organism, as well as for somatic gene therapy.
BACKGROUND OF THE INVENTION
It is known that proteins for technical application or for therapeutic purposes can be expressed in sufficient quantity by the transfer of genes in microorganisms or mammalian cells. These procedures are particularly important for proteins occurring naturally in the body, such as hormones, regulatory factors, enzymes, enzyme inhibitors and humanized monoclonal antibodies which are otherwise only available to a limited extent or not available at all. The procedures are also important for producing surface proteins of pathogenic microorganisms or viral envelope proteins so as to safely produce diagnostic tests and for the development of efficacious vaccines. Through protein engineering it is also possible to produce new types of proteins, which through fusion, mutation or deletion of the corresponding DNA sequences, have properties optimized for particular uses, for example immunotoxins.
Genes obtained from human cells are also functional in mouse, rat or sheep cells and there lead to the formation of corresponding gene products. This has already been made use of in the production of therapeutic products, for example in the milk of transgenic farm animals. The hitherto known method has been by the microinjection of corresponding foreign DNA carrying vectors into the nucleus of the fertilized egg cell, in which the DNA is then incorporated into the chromosome with a yield of 1%. The transgenic fertilized egg cell is then transplanted into hormonally stimulated mother animals. An offspring carrying the transfected gene in all its body cells is the basis for the creation of a “transgenic herd/flock”. Using gene technology it is now possible to alter farm animals in such a targeted way that they produce human proteins in their blood, tissue or milk, which cannot be produced by microorganisms or plants.
However, the use of transgenic animals as protein production factories has the decisive disadvantage that it is necessary to manipulate the germ line of the animal. Due is to the considerable expenditure of technology and time required to create and breed transgenic animals and also due to the discussions regarding the ethical consequences of these methods, alternative methods for producing proteins in animal hosts without manipulation of the germ line are necessary and would be very advantageous.
It is known, furthermore, that the milk of mammals such as cows, sheep, goats, horses or pigs can contain a range of disease-causing bacterial agents. Among such agents are
Listeria, mycobacteria, Brucella, Rhodococcus, Salmonella, Shigella, Escherichia, Aeromonads
and
Yersinia
or general bacteria with intracellular lifestyle [1, 2]. These bacteria are usually transmitted to humans or animals through oral ingestion [3], but can also be transmitted by droplet infection. A major source for the infection of humans with
Listeria
[4], mycobacteria [5] and
Escherichia coli
is contaminated milk [6]. Humans ingest the bacteria when consuming unpasteurised milk or milk products. The other bacteria types listed above, such as Salmonella, Shigella, Yersinia, Rhodococcus and Brucella are transmitted to humans in a similar way. However, bacteria may also enter humans through other bacterially infected animal products from cows, goats, sheep, hares, horses, pigs or poultry.
The infection of animals frequently occurs through mucosal surfaces and very frequently through the digestive tract. However, after ingestion of bacteria, for example in the case of
Listeria
, not all tissues show symptoms of infection. In cows and goats the infection is mainly evident in the udder, spleen and liver. In sheep there may additionally be illness in the central nervous system in the form of meningitis, so not all animals survive the infection. With infection of the udder, the infection chain is closed. With contaminated milk, acquired bacteria can reinfect another animal, for example a suckling calf or a human via the digestive tract.
The following is known at present regarding the process of bacterial infection in humans, here presented using the example of
Listeria
:
Of the six known
Listeria
species, only
L.monocytogenes
and
L.ivanovii
are pathogenic for humans [7]. Illness in humans results from consuming infected milk or milk products. The course of the illness depends on the state of health of the individual and is generally inapparent. Intrauterine transmission of bacteria to the fetus may occur during pregnancy, resulting in abortion, stillbirth or premature birth. In all cases excellent and problem-free treatment exists using antibiotics such as ampicillin or erythromycin [8; 8a].
The mode of entry into the cell occurs is well defined for
L.monocytogenes
in humans and animals and for
L.ivanovii
in sheep. For full pathogenicity of
Listeria
to occur, a range of pathogenicity factors are necessary. Among them are PrfA (positive regulator of virulence), ActA (actin nucleating protein), PlcA (phosphatidylinositol-specific phopholipase), PlcB (phosphatidylcholine-specific phopholipase), Hly (listeriolysin), Mpl (metalloprotease) [9]. The cell specificity of the pathogen—host cell interaction is mediated through a range of proteins. Among these are the internalins InlA and InlB, which are involved in the initial contact and the interaction of bacteria and cell surface [10, 11]. Under experimental conditions
L.monocytogenes
can also infect endothelial cells, epithelial cells, fibroblasts and hepatocytes. In addition,
L.monocytogenes
can infect cells of the white blood cell count like neutrophilic granulocytes, macrophages and lymphocytes. This is a significant factor in the transmission of bacteria from the site of primary infection to the target organ in the host. Finally, lung tissue can also be infected by
Listeria
if the bacteria are applied as a droplet infection.
After adhering to the cell surface,
L.monocytogenes
is taken up by the cell by endocytosis, the bacterium breaks down the endosome membrane under the effect of listeriolysin (Hly) and is thus released into the cell cytosol [14]. Once inside the cell, the bacteria can proliferate. With the production of further proteins, the fully pathogenic bacteria does not stay localized but actively spreads to distal sites . Bacterial spread is effected by using a range of proteins from
L.monocytogenes
itself and some cellular proteins [15, 16]. ActA is expressed on the cell surface of
L.monocytogenes
. It binds the cellular protein VASP, which for its part forms the bridge required for the attachment of cellular actin. Actin tails subsequently develop, which carry the bacterium at their tip and thus move it further through the cell. If
L.monocytogenes
contacts the cell membrane, a membrane protrusion forms, which projects directly into any adjacent cells if they are present. This protrusion is then endocytosed by the adjacent cell so the
L.monocytogenes
is then inside the new cell within a double membrane. The two membranes are dissolved under the effect of Hly and PlcB [171]. At the end of this process
L.monocytogenes
has also infected the neighbouring cell and the infection process begins again. In this way
L.monocytogenes
enters, for example, secretory cells of the cow udder. Secreted
Listeria
proteins are detectable in milk, i.e. they are passed on intracellularly from the lactating cell into the milk [18]. Hly (listeriolysin) and IrpA (internalin related protein [19]) are two pathogenicity factors belonging to this group of proteins which are produced, secreted and passed out in milk in large quantities by
L.monocytogenes
[20].
Knowledge of the infection process has made it possible to al

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