Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1994-11-01
2002-06-11
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S192100, C530S395000, C514S569000, C435S007320, C435S012000
Reexamination Certificate
active
06403099
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to conjugated compounds consisting of heat shock proteins and polysaccharides or oligosaccharides, in particular those polysacccharides or oligosaccharides derived from the capsule of pathogenic microorganisms. Such compounds are capable of inducing the formation of anti-polysaccharide antibodies and are accordingly useful as vaccines for use in man and in animals.
STATE OF THE ART
Bacteria are the aetiological agents for a wide range of disease conditions.
Examples of such diseases include meningitis caused by
Neisseria meningitidis
and other infections caused by
Haemophilus influenzae
Type b (Hib) or Streptococcus (including Pneumococcus), typhoid fever caused by infection with
Salmonella typhi
, intestinal disease caused by non-typoidal Salmonella or Shigella bacteria.
It is known that protective immunity to capsular bacteria is mediated by antibodies to the capsular polysaccharides. It is also known that, in order to obtain sufficient stimulation of the immune system, it is necessary to conjugate capsular polysaccharides to carrier proteins (Robbins et al, J. Infect. Dis., 1990, 161,821-832).
In particular, there have been described in the literature conjugated compounds consisting of polysaccharides (for example Group C meningococcal polysaccharide (MenC), Hib and Group A meningococcal polysaccharide (MenA)) and proteins such as CRM-197 (a peptide derived from
Corynebacterium diphtheriae
), TD (Diphtheria toxoid) or TT (Tetanus toxoid—see Peeters et al. Inf.Immun., (October 1991), 3504-3510; Claesson et al., J. Pediatrics St Louis, 112(5), 695-702, (May 1988).
Some such vaccines are already used with good results in clinical practice. However, there exists the need to identify novel protein carriers which impart to the conjugates immunogenic properties better than those achieved with the carriers used hitherto.
The present invention relates to the use of heat shock proteins as a protein carrier to increase the immunogenic response of oligosaccharides and polysaccharides.
Heat shock proteins are known to contain a significant number of T epitopes and thus to stimulate the cellular immune system.
A conjugated compound of the heat shock protein of
Mycobacterium bovis
(65 kDa), as a carrier for a malarial epitope, has been described as inducing a marked immunity in animals pre-immunised with Bacillus Calmette-Guérin (BCG) without requiring adjuvants (Lussow et al Eur. J. Immunol., 1991, 21,2297-2302). It is however to be noted that the effects observed in Lussow et al relate to T cell dependent effects exhibited by peptides (which are well known to be T-cell dependent) conjugated to heat shock proteins.
More particularly, because the heat shock proteins are well conserved across bacteria of different strains and type, adventitious infection with bacteria, which is a continuous process, will ensure that the immune system remains sensitised to heat shock proteins, thus ensuring a good response to the conjugate compounds of the invention either at primary vaccination or on administration of a booster vaccination.
The present invention permits the use of bacterial capsular polysaccharides and oligosaccharides to be used without adjuvants (although adjuvants can be used).
Large numbers of children are given BCG vaccine (which will include bacterial heat shock proteins) to guard against tuberculosis and therefore a conjugate of the present invention containing a heat shock protein as the carrier would find a large number of subjects already pre-immunised with the carrier precisely as a result of the BCG vaccination which they have undergone.
Again, since the heat shock proteins are highly conserved even the population which has not been vaccinated with BCG can easily develop immunity (as a result of natural interaction with other bacteria) and can hence find itself in a state of being able to develop a good immune response following vaccination with a conjugate composed of a heat shock protein and a T cell-independent antigen (oligosaccharide or polysaccharide). Thus the carriers of the present invention uniquely exploit the high conservation of heat shock proteins across bacteria and T-cell memory to ensure high titre vaccination.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a conjugate compound comprising at least one heat shock protein or portion thereof including at least one immunostimulatory domain and at least one oligosaccharide or polysaccharide.
The heat shock protein may be any heat shock protein capable of exhibiting an immunostimulatory effect in animals, preferably humans.
The heat shock proteins are highly conserved in bacteria, parasites and mammals. Any heat shock protein can be used in the conjugates of the present invention, provided it exhibits a positive immunostimulatory effect in the intended immunisation subject without significant deleterious effects. Specific, non-limiting examples include heat shock proteins from
Helicobacter pylori, P. aeruginosa, C. trachomatis
and
M. leprae
, especially the hsp60 group of heat shock proteins.
More particularly, three heat shock proteins are specifically exemplified herein, namely,
M. bovis
BCG GroEL-type 65 kDa hsp (hspR65), Recombinant
M.tuberculosis
DnaK-type 70 kDa hsp (hspR70) and a novel heat shock protein from
H.pylori.
The
H. pylori
heat shock protein (hsp) is a protein whose nucleotide and amino acid sequence is given in FIG.
3
and whose molecular weight is in the range of 54-62 kDa, preferably about 58-60 kDa. This hsp belongs to the group of Gram negative bacteria heat shock proteins, hsp60. In general, hsp are among the most conserved proteins in all living organisms, either prokaryotic and eukaroytic, animals and plants, and the conservation is spread along the whole sequence.
The conjugate may contain one or more heat shock proteins or immunostimulatory domains thereof. The heat shock proteins may the same or different. Preferably however, one heat shock protein or a portion containing one or more immunostimulatory domains is present.
As used herein, the term “immunostimulatory domain” refers to a region of a heat shock protein amino acid sequence capable of enhancing the immune reaction of a subject mammal to a polysaccharide or oligosaccharide component of a conjugate compound including the domain.
An advantage of using only specific domains from complete heat shock proteins is that it is possible selectively not to include domains common to human heat shock proteins. For human vaccination this is advantageous as such regions will not affect the immunostimulatory effect of the heat shock protein as they will be recognized as “self”. In addition any immunity that is stimulated against such “self” regions might lead to autoimmunity.
Suitable domains of the hsp60 family of heat shock protein are identified in
FIG. 2
by underlining of sequence of reduced homology with the human heat shock protein. Functional sub domains within the domains shown in
FIG. 2
(SEQ. ID NO: 1); (SEQ. ID NO: 2); (SEQ. ID NO: 3); (SEQ. ID NO: 4); and (SEQ. ID NO: 5) may also be used, as can domain and sub domain combinations.
The skilled man can readily ascertain for a given heat shock protein which domains or epitopes are responsible for the immunostimulatory action and prepare modified heat shock protein containing only those domains or a sub set thereof.
The oligosaccharide or polysaccharide component of the conjugate compound may be the complete capsular polysaccharide or oligosaccharide of any pathogenic microorganism against which vaccination is indicated or a portion thereof capable of eliciting protective immunity. The oligosaccharide or polysaccharide may be from a single bacteria or from two or more bacteria.
Particular non-limiting examples of bacteria which may be targeted include:
Haemophilus influenzae
Type b (Hib), Streptococcus (including pneumococcus), Salmonella especially
Salmonella typhi
, intestinal disease caused by non-typoidal Salmonella or Shigella bacteria.
According to a particular embodiment
Costantino Paolo
Norelli Francesco
Rappuoli Rino
Viti Stefano
Attwell Gwilym J.O.
Blackburn Robert P.
Chiron S.p.A.
Harbin Alisa A.
Portner Ginny Allen
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