Title:  Two-phase system for the production and presentation of foreign antigens in hybrid live vaccines

United States Patent:  6,255,097

Assignee:  Max-Planck-Gesellschaft Zu Forderung der Wessenchaften (Gottingen, DE)

Filed:  November 22, 1993

PCT NO:  PCT/EP91/02478

102(e) Date:  November 22, 1993

PCT PUB. Date:  July 9, 1992

Foreign Application Priority Data:  Dec 20, 1990[DE] (40 41 045)

The present invention relates to a genetic engineering process for the optimal production and exposure to the immune system of additional antigen coded for by a live vaccine. The genetic engineering process is based on the use of spontaneous DNA reorganisation in the recombinant live vaccine, such that the recombinant live vaccine spontaneously divides into two subpopulations (A and B), whereby subpopulation A is capable of infecting and acts immunogenically per se as a minimum characteristic and subpopulation B as a minimum characteristic is regenerated by subpopulation A, produces additional antigen and acts immunogenically with respect to said additional antigen. The formation of two subpopulations of the live vaccine ensures, on the one hand, that the infection process necessary for the induction of an immune response takes place and, on the other hand, that the formation of additional antigen by a hybrid live vaccine does not disturb the infection process in order to finally achieve an effective immune response to the additional antigen and the pathogen cross-reacting therewith.

DETAILED DESCRIPTION OF THE INVENTION

Random events which lead to the formation of subpopulations are natural and can mostly be traced back to changes in the DNA, so-called (programmed) DNA reorganisations (Borst, Science, 235:658-667, 1987). In principle, all naturally-observed mechanisms of DNA reorganisation can be used for the task set, provided that they can be reproduced suitably frequently in the hybrid live vaccine. The frequency of the formation of subpopulation B is preferably 0,1% to 50% per cell and cell generation; in particular cases, the frequency can, however, be higher or lower. Particularly suitable for application in a hybrid live vaccine are simple mechanisms of DNA reorganisation which occur at specific sites, such as inversion (Craig, Cell, 41:649-650, 1985) or deletion by resolution of transposon cointegration (Grindley, Annual Rev. Biochem., 54:863-896, 1985) of a DNA segment. However, other site-specific DNA reorganisations or such DNA reorganisations which are based on slipped-strand-mispairing (Levinson, Mol. Biol. Evol. 4:203-221, 1987; Stern, Cell, 47:61-71, 1986) seem suitable for the cited object.

It must be the purpose of the cited spontaneously occurring DNA reorganisation in a live vaccine to lead directly or indirectly to the production of additional antigen, i.e. to the formation of subpopulation B which produces the antigen. This occurs very simply e.g. by positioning an expression signal (for example the promoter) of a gene by DNA reorganisation in front of the gene such that said gene changes from a non-expressed to an expressed state. All variants of this principle are possible; but they all have the goal of bringing about a change in the expression of a gene by DNA reorganisation. It usually makes sense to switch on genes by DNA reorganisation although the opposite is also possible.

The gene switched on by DNA reorganisation can, on the one hand (model 1), be the gene coding for an additional antigen or (if the additional gene is not a protein but an enzymatic synthesis product, e.g. a carbohydrate) a gene required for the antigen synthesis, or, on the other hand (model II), a gene encoding a protein which controls the expression of the actual gene coding for the antigen. Model I is thus a system which by DNA reorganisation directly codes for the synthesis of the additional antigen or for an enzyme required for the synthesis while model II represents a system which allows the production of the additional antigen via a cascade system. The cascade system can be realized e.g. in that the gene directly controlled by DNA reorganisation codes for an RNA polymerase which is specific for the promoter preceding the gene coding for the antigen, or a gene regulator which in another specific manner induces the expression of the gene coding for the antigen (e.g. T7 polymerase: Studier, Meth. Enzymol. 185:60-89, 1990; lac system: De Boer, Proc. Natl. Acad. Sci. USA, 80:21-25, 1983). In this case too, there are all the possibilities of variation which nature offers. Whilst model I is on the one hand less complicated, the application of model II has advantages because high levels of expression can be achieved after one single DNA reorganisation due to the increasing effect of the cascade. Furthermore, with this model several genes which code for different additional antigens within one hybrid live vaccine can be switched on at the same time.

The realization of the described system is technically particularly simple in bacterial live vaccines. The genetic element capable of DNA reorganisation can be held in bacteria e.g. on a plasmid or introduced in the genome by means of a phage, a transposon or by homologous recombination. In the case of the cascade model II, the antigen-encoding genes, which have special sites for the binding of the gene products of the element capable of DNA reorganisation, can be introduced in a similar manner by using conventional techniques.

Depending on the principle of the underlying DNA reorganisation enzymes (referred to here as "control enzymes") are necessary which have to be provided by the cell so that DNA reorganisation can take place at all. In the case of an inversion according to the principle of the phage Mu an invertase is necessary, for example, besides cellular factors (Kahmann, Cell, 41:771-780, 1985); in the case of a deletion corresponding to the mechanism of the resolution of transposon cointegration the enzyme resolvase is necessary, for example, (Reed, Cell, 25:713-719, 1981); the formation of replicative circles corresponding to the replication of filamentous phages requires inter alia the gene 2 product of the phage (Meyer, Nature, 296:828-832, 1982). These enzymes as well as the DNA structure at which they attack (referred to here as "target sites"; e.g.: Mertens, EMBO J., 7:1219-1227, 1988), offer a suitable basis from which to regulate the frequency with which DNA reorganisation forms the additional antigen and thus to determine the ratio of populations A and B. Manipulation of the expression of the control enzymes or the target sites by genetic engineering makes it possible to adjust this ratio exactly to the desired ratio of the populations. However, it is also conceivable to subject the control enzymes in turn to a superordinate regulatory control in order to change the ratio of populations A and B by external influences (e.g. in dependence on the temperature). Here, too, there are possibilities of variation as desired and given in the state of molecular biology.

The conventional methods of molecular biology serve for the construction of genetic elements which undergo DNA reorganisation. Moreover, it is advantageous to firstly clone such genetic elements in cells which do not synthesise control enzymes in order to keep them stable for manipulation and construction purposes. After preparation such elements can be inserted into the genome of the live vaccine by the conventional methods of molecular biology or can be kept extrachromosomally as a plasmid. The same applies to the genes which code for the control enzymes as well as with respect to genes indirectly or directly coding for antigens in the case of model II.

The final hybrid live vaccine is administered in a suitable manner (e.g. by oral dosage or by injection) to the individual to be protected. The administered dose of the hybrid live vaccine usually corresponds to that for the corresponding non-hybrid live vaccine.

Claim 1 of 25 Claims

What is claimed is:

1. An immunogenic composition comprising a living cell or a virus expressing a first immunogenic antigen and further comprising recombinant DNA encoding at least one second, immunogenic antigen heterologous to said cell or virus, wherein said at least one second antigen is expressed under the control of a DNA reorganization occurring in said cell or in a cell infected with said virus, the DNA reorganization resulting in a phase variation in the cell or virus, wherein the DNA reorganization occurs in a subject to which said living cell or virus is administered.

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