Transferrin receptor subunit proteins of Neisseria meningitidis

Details Transferrin receptor subunit proteins of Neisseria meningitidis Transferrin receptor subunit proteins of Neisseria meningitidis

1997-06-03

2001-12-04

Kemmerer, Elizabeth (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C435S069100

Reexamination Certificate

active

06326350

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The subject of the present invention is DNA fragments of
Neisseria meningitidis
which encodes the transferrin receptor subunits as well as a process for producing each of the subunits by the recombinant route.
Meningitides are generally either of viral origin or of bacterial origin. The bacteria mainly responsible are:
N. meningitidis
and
Haemophilus influenzae
, which re respectively implicated in about 40 and 50% of cases of bacterial meningitides.
In France, about 600 to 800 cases of
N. meningitidismeningitides
are recorded per year. In the United tates, the number of cases is up to about 2,500 to 3,000 per year.
The N. meningitidis species is sub-divided into serogroups according to the nature of the capsular polysaccharides. Although about twelve serogroups exist, 90% of meningitis cases can be attributed to 3 sero-groups: A, B and C.
2. Description of the Related Art
Effective vaccines based on capsular polysaccharides exist for the prevention of meningitides caused by
N. meningitidis
serogroups A and C. These polysaccharides as they are only slightly or not at all immunogenic in children below 2 years and do not induce immunological memory. However, these disadvantages can be overcome by conjugating these polysaccharides with a carrier protein.
In contrast, the polysaccharide of
N. meningitidis
group B is not at all or is only slightly immunogenic in man whether it is in conjugated form or not. Thus it appears highly desirable to seek out a vaccine against meningitides induced by
N. meningitidis
especially of the serogroup B other than a polysaccharide-based vaccine.
To this end, various proteins of the outer membrane of
N. meningitidis
have already-been proposed. These are in particular the membrane receptor for human transferrin.
In general, the great majority of bacteria require iron for their growth and they have developed specific systems for acquiring this metal. With regard especially to
N. meningitidis
which is a strict pathogen for man, the iron can only be derived from human iron transport proteins such as transferrin and lactoferrin since the quantity of iron in free form is negligible in man (of the order of 10
−18
M), in any case insufficient to permit bacterial growth.
Thus,
N. meningitidis
has a human transferrin receptor and a human lactoferrin receptor which enable it to bind these iron-chelating proteins and subsequently to capture the iron required for its growth.
The transferrin receptor of the strain B16B6 of
N. meningitidis
has been purified by Schryvers et al. (WO 90/12591) from a membrane extract. This protein, as purified, appears to consist essentially of 2 types of polypeptides: a polypeptide with a high apparent molecular weight of 100 kD and a polypeptide with a lower apparent molecular weight of about 70 kD, as visualised after SDS-polyacrylamide gel electrophoresis.
The purification product especially identified by Schryvers is by arbitrary definition and for the purposes of the present patent application, called transferrin receptor and its constituent polypeptides, subunits. In the text which follows, the subunits of high molecular weight and of lower molecular weight are called Tbp1 and Tbp2 respectively.
However, the purification process described by Schryvers et al. cannot be used for the large-scale production of the transferrin receptor. The industrial preparation of this receptor in purified form necessarily involves a production step using a heterologous expression system.
SUMMARY OF THE INVENTION
To this end, the object of the invention is to provide DNA fragments which encode the transferrin receptor subunits of
N. meningitidis.
Moreover, since the pioneering work of Schryvers et al., it has been discovered that there are in fact at least 2 types of strains which differ by the constitution of their respective transferrin receptors. This was demonstrated by studying membrane extracts of several tens of
N. meningitidis
strains of diverse origins. These membrane extracts were first subjected to an SDS-polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose membranes. These nitrocellulose membranes were incubated:
a) in the presence of a rabbit antiserum directed against the transferrin receptor purified from the strain B16B6 of
N. meningitidis
, also called IM2394;
b) in the presence of a rabbit antiserum directed against the transferrin receptor purified from the strain IM2169 of
N. meningitidis
; or
c) in the presence of peroxydase-conjugated human transferrin.
With regard to a) and b), the recognition of the transferrin receptor subunits is visualised by the addition of a peroxydase-coupled anti-rabbit immunoglobulin antibody and then by the addition of the substrate for this enzyme.
Tables I and II below show the profile of some representative strains as it appears on a 7.5% polyacrylamide gel after SDS gel electrophoresis; the bands are characterised by their apparent molecular weights expressed in kilodaltons (kD):
TABLE I
Strains
2394 (B; 2a;P1.2:L2,3)
2234 (Y; nd)
2228 (B; nd)
2154 (C; nd)
550 (C; 2a:)
2170 (B; 2a:P1.2:L3)
2448 (B; nd)
179 (C; 2a:P1.2)
Detection
93
93
99
with
anti-2394
receptor
antiserum
68
69
69
Detection
93
93
99
with
anti-2169
receptor
antiserum
Detection
68
69
69
with
transferrin-
peroxydase
N.B. In brackets are indicated in order the serogroup, the serotype, the subtype and the immunotype.
TABLE II
Strains
2169
1000
1604
132
1001
876
1951
2449
867
(B:9:P1.9)
(B:nd)
(B:nd)
(C:15:P:1.16)
(A:4:P1.9)
(B:19:P1.6)
(A:nd)
(B:nd)
(B:2b:P1.2)
Detection with anti-2394
96
98
98
98
98
96
94
94
93
receptor antiserum
Detection with anti-2169
96
98
98
98
98
96
94
94
93
receptor antiserum
87
85
83
81
79
88
81
85
85
Detection with traneferrin-
87
85
83
81
79
88
87
85
85
peroxydase
N.B. In brackets are indicated in order the serogroup, the serotype, the subtype and the immunotype.
The results entered in the first 2 lines of the tables show that there are 2 types of strains:
The first type (Table I) corresponds to strains which possess a receptor whose 2 subunits, under the experimental conditions used, are recognised by the anti-IM2394 receptor antiserum whereas only the high molecular weight subunit is recognised by the anti-IM2169 receptor antiserum.
The second type (Table II) corresponds to strains which possess a receptor whose 2 subunits, under the experimental conditions used, are recognised by the anti-I2169 receptor antiserum whereas only the high molecular weight subunit is recognised by the anti-IM2394 receptor antiserum.
Consequently, an antigenic diversity exists at the level of the subunit of lower molecular weight. This diversity is however limited since it is of 2 main types, contrary to what is suggested by Griffiths et al., FEMS Microbiol. Lett. (1990) 69:31.
By virtue of these observations, it could have been supposed. that an effective vaccine against all
N. meningitidis
infections could be adequately made up of the high molecular weight subunit, irrespective of the strain from which the receptor originates, since the said subunit is recognised by the 2 types of antisera. However, it appears that this cannot be the case since the high molecular weight subunit is thought-to be incapable of inducing the production of neutralising type anti-bodies. Only the smallest of the 2 receptor subunits is thought to be capable of performing this function. Since this subunit of lower molecular weight is characterised by a significant antigenic variation from the first type to the second type of strain, a single type of transferrin receptor could not be sufficient for vaccinating against all
N. meningitidis
infections. Consequently, a vaccine should contain at least the subunit of lower molecular weight of each of the strains IM2394 and IM2169 or their respective equivalents and, optionally, the high molecular weight subunit of at least one
N. meningitidis
strain.


REFERENCES:
patent: 5141743 (1992-08-01), Schryvers
patent: 5618540 (1997-04-01), Quentin-Millet
patent: 5618541 (1997-04-01), Que

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