Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1999-09-16
2003-12-09
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093200, C424S200100, C424S203100, C424S184100, C424S254100, C424S234100, C424S242100
Reexamination Certificate
active
06660261
ABSTRACT:
BACKGROUND OF THE INVENTION
Pertussis
, the most responsible agent of which is the gram-negative bacterium
Bordetella pertussis
, remains a current infectious disease throughout the world. More than 50 millions cases are declared each year and a mortality estimation is 500 000 deaths per year, mainly infants. Mortality is for example high in the developing countries where vaccination is not sufficient and
pertussis
represents one of the major causes of infantile mortality. The advent of cellular vaccines consisting in chemically or thermally inactivated
B. pertussis
has led to a spectacular reduction of
pertussis
occurrence. However, limitations related to their use have appeared during these last two decades. Various reports have thus put forward the hypothesis of a relationship between the administration of the cellular vaccine against
pertussis
and some serious side-effects.
Moreover, the need to administrate various vaccinal doses to obtain a maximum protection, the variability of efficiency of the different batches of vaccines and the resurgence of the disease in adults due to a weakening of post-vaccinal immunity during the period of the post-vaccinal immunity in the absence of late re-vaccinations are as many inconveniences that have led to the development of cellular vaccines. However, the protective antigens comprising those new generation vaccines, generally defined as more efficient, more secure and more immunogenic than cellular vaccines, have not been clearly defined yet and multiple injections are still necessary. Moreover, serological correlations with the protection of children have not still been determined. Finally, the production cost of these vaccines has been found higher than this of the prior ones, which represents an important problem for developing countries that are most demanding.
On the other hand, it has been shown that the natural infection with
B. pertusiss
leads to a long-term protection, whereas the one induced by vaccination had a limited time (Bass, J. W. et al. 1987 Pediatr. Infect. Dis. J. 6:141-144, Jenkinson, D. 1988, Br. Med. J. 296:612-614). The reasons for this difference are not clear, showing the lack of knowledge relating to immunizing mechanisms involved in each case. One of the potential explanations is based upon a better induction of the immunologic memory at the respiratory tract after an infection, compared to the one obtained after a parenteral immunization. Moreover, a Th1-type cellular response T, which could play an important part in the protective immunity, is induced through the natural infection, whereas immunization with the cellular vaccine shows rather a strong response Th2 (Mills, K. H. G. et al. 1983, J. Med. Microbiol. 39:163-164). Finally, the infection stimulates IgA production against
B. pertussis
antigens in the serum and the secretions, whereas these antibodies are not frequently detected on vaccinated individuals (Shahin, R. et al. 1992, in J. E. Ciardi et al. Eds Genetically Engineered Vaccines, Plenum Press N.Y.).
All those observations led to the belief that a long-term protection against
pertussis
would be better obtained through a vaccination with attenuated living bacteria administrated at the level of the respiratory mucosas, which are the natural openings during the infection by
B. pertussis
. Prior work has shown that immunization of mice either with spontaneous attenuated living strains (Vesselinova-Jenkins, C. K., 1985. Dev. Biol. Stand. 61:517-524) or aroA strains (Roberts, F. et al. 1990. Infect. Immun. 58:732-739) of
B. pertussis
protects against a second infection. However, such strains are inconvenient in that they are badly characterized and hold a certain toxicity, or have lost to a great extent their colonizing capacity, and thus require numerous administrations to induce an efficient protective immunity.
The
B. pertussis
toxin is an oligomer protein formed with five sub-units so-called S1 to S5. It may be shared into two great domains so-called protomer A (consisting in S1) and oligomer B (consisting in S2 to S5). The oligomer B is responsible for the toxin interaction with its receptors at the surface of the target cells and the protomer A (or S1) is injected into the target cell and expresses therein an ADP-ribosyltransferase enzyme activity (Tamura et al., Biochemistry 21, 5516-5522, 1962). The genes coding for the five sub-units have been cloned and sequenced (Locht and Keith, Science 232, 1258-1264, 1986) and the crystal structure has been established (Stein et al., Structure 2, 45-47, 1994). Various mutations in the toxin gene have been described as being responsible for the genetic inactivation of this molecule. These mutations may be found in S1 gene (review, see Locht and Antoine, Biochimie 77, 333-340, 1995) and/or in the genes coding for the oligomer B (see for example Lobet et al. J. Exp. Med. 177, 79-87, 1993). The toxin may be put in evidence through an immunologic activity with polyclonal or monoclonal antibodies, such as for example the antibody lB7 mentioned thereafter or through its biological activity, such as the activity on the cells “Chinese Hamster Ovary” as described by Hewlett et al. (Infect. Immun. 40, 1198-1203, 1983).
Filamentous hemagglutinin (FHA) is the major adhesion produced and secreted by
B. pertussis
(review, see Locht et al. Mol. Microbiol., 9, 653-660, 1993). The structure gene of the FHA, so-called fhaB, has been clond (Brown, D. R. et al. 1987. Infect. Immun. 55:154-161; Relman, D. A. et al. 1989. Proc. Natl. Acad. Sci. USA. 86:2637-2641; Delisse-Gathoye, A.M. et al. 1990. Infect. Immun. 58:2895-2905) and codes for a precursor (FHAB) of about 367 kDa. The mature form (220 kDa) corresponds to the N-terminal two thirds of FHAB and has a structure so-called “hair pin” (Makhov et al., J. Mol. Biol. 241, 110-124, 1994). Although the C-terminal part of FHAB is not present under the mature form, it has interesting characteristics such as various regions known to be rich in proline and an RGD site. Moreover, this C-terminal part of the precursor appears to play an important part in FHA secretion. Downwards the N-terminal region of the FHA two repetitive regions, respectively A and B, are located, followed by a proteolysis-sensitive site and by a RGD sequence. The FHAB maturation site is located at about 1000 amino-acids downwards this RGD sequence. The FHAB N-terminal domain plays an essential part in the secretion of the mature protein since a phase deletion of this region appears to inhibit totally the FHA biogenesis. The FHA secretion depends upon a minor protein so-called-FhaC present at the level of the external membrane of
B. pertussis
. This protein is coded by the gene FhaC located downwards FhaB and separated from this latter by three other genes, fimB, fimC and fimD, coding for the fimbriae. An FHA secretion mechanism involving cleavage and then modification of the N-terminal part of FHAB, followed by interaction between this region and the minor protein FHAC, have been proposed.
The various FHA binding mechanisms allow the
B. pertussis
to adhere to numerous types of eukaryote cells. Although the
B. pertussis
toxin is involved in the adherence of the bacterium with the epithelial cells of the respiratory tract, the FHA plays a major part in such an interaction. The FHA can interact with glycoconjugates and the carbohydrate recognition domain has been identified in the FHA region limited by the amino-acids 1141 to 1279 which would correspond to the buckle of the “hair-pin” structure which already contains the RGB site in positions 1097-1099. The FHA is also the major adhesin involved in the adherence mechanism of
B. pertussis
with the unciliated epithelial cells. An activity of binding this molecule to the heparin sulphated glycosaminoglycan, present in non negligible amount in the bronchial mucus as well as in the extracellular matrix and at the surface of numerous epithelial cells is responsible for the bacterium adherence with these cells. Recent studies make obvious a specific heparin binding site located in the N-
Capron Andre
Locht Camille
Mielcarek Nathalie
Poulain-Godefroy Odile
Riveau Gilles
Devi S.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur de Lille
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