Recombinant Haemophilus influenzae adhesin proteins

Details Recombinant Haemophilus influenzae adhesin proteins Recombinant Haemophilus influenzae adhesin proteins

1999-03-16

2002-01-01

Graser, Jennifer E. (Department: 1641)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C536S023100, C536S023700, C536S024100, C536S024330, C435S069100, C435S071100, C435S252300, C435S320100, C435S243000, C424S256100, C424S200100

Reexamination Certificate

active

06335182

ABSTRACT:

FIELD OF INVENTION
The present invention relates to the field of molecular genetics and, in particular, to the production of recombinant
Haemophilus influenzae
adhesin (Hia) proteins.
BACKGROUND TO THE INVENTION
Haemophilus influenzae
is the cause of several serious human diseases, such as meningitis, epiglottitis, septicemia and otitis media. There are six serotypes of
H. influenzae
, designated a to f, that are identified by their capsular polysaccharide.
H. influenzae
type b (Hib) was a major cause of bacterial meningitis until the introduction of several Hib conjugate vaccines in the 1980's (ref. 1. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Vaccines based upon
H. influenzae
type b capsular polysaccharide conjugated to diphtheria toxoid (ref. 2), tetanus toxoid (ref. 3 and U.S. Pat. No. 4,496,538), or
Neisseria meningitidis
outer membrane protein (ref. 4) have been effective in reducing
H. influenzae
type b-induced meningitis. The other serotypes of
H. influenzae
are associated with invasive disease at low frequencies, although there appears to be an increase in the incidence in disease caused by these strains as the incidence of Hib disease declines (ref. 5; ref. 6). Non-encapsulated or non-typeable
H. influenzae
(NTHi) are also responsible for a wide range of human diseases including otitis media, epiglottitis, pneumonia, and tracheobronchitis. The incidence of NTHi-induced disease has not been affected by the introduction of the Hib vaccines (ref. 7).
Otitis media is the most common illness of early childhood, with 60 to 70% of all children, of less than 2 years of age, experiencing between one and three ear infections (ref. 8). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children.
H. influenzae
infections account for about 30% of the cases of acute otitis media and about 60% of chronic otitis media. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. Furthermore, many of the causative organisms of otitis media are becoming resistant to antibiotic treatment. An effective prophylactic vaccine against otitis media is thus desirable.
During natural infection by NTHi, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and, therefore, potential vaccine candidates. A family of high molecular weight proteins (HMW1 and HMW2) that are important in attachment of NTHi to epithelial cells has been identified in about 70 to 75% of NTHi strains (ref. 9; ref. 10). These high molecular weight adhesins have been shown to afford some protection in the chinchilla model of otitis media (ref. 11). A second family of high molecular weight adhesion proteins has been identified in about 25% of NTHi and in encapsulated
H. influenzae
strains (ref. 12; ref. 13, ref. 14). The NTHi member of this second family is termed
Haemophilus influenzae
adhesin or Hia and the homologous protein found in encapsulated strains is termed
Haemophilus influenzae
surface fibril protein or Hsf. The hia gene was originally cloned from an expression library using convalescent sera from an otitis media patient, which indicates that it is an important immunogen during disease. The prototype Hia and Hsf proteins demonstrate about 82% sequence similarity, although the Hsf protein is considerably larger. The proteins are comprised of conserved amino and carboxy termini and several repeat motifs, with Hsf containing more repeat sequences than Hia. A high molecular weight protein (200 kDa) has also been identified from Moraxella catarrhalis that has some sequence homology with the Hsf and Hia proteins (U.S. Pat. No. 5,808,024).
Since Hia or Hsf is conserved amongst encapsulated strains of
Haemophilus influenzae
and about 20 to 25% of non-encapsulated strains, and has been demonstrated to be an adhesin, the protein has utility in diagnosis of and vaccination against disease caused by
H. influenzae
or other bacterial pathogens that produce Hia or a protein capable of raising antibodies specifically reactive with Hia.
A disadvantage of Hia for use as an antigen in diagnosis, for the generation of anti-Hia antibodies useful in diagnosis and as an immunogen in vaccination is the low recovery of the native protein from
Haemophilus influenzae
species.
It would be advantageous to provide recombinant Hia protein for use as antigens, in immunogenic preparations including vaccines, carriers for other immunogens and in the generation of diagnostic reagents.
SUMMARY OF THE INVENTION
The present invention is directed towards the provision of recombinant
H. influenzae
adhesin (rHia) proteins.
In connection with the provision of such recombinant proteins, the present invention provides certain isolated and purified nucleic acid molecules. Accordingly, in one aspect thereof the present invention provides an isolated and purified nucleic acid molecule encoding a
Haemophilus influenzae
adhesin (Hia) protein of a strain of
Haemophilus influenzae
having: (a) a DNA sequence selected from the group consisting of those shown in
FIGS. 18
,
19
,
20
,
21
,
22
,
23
,
24
and
25
(SEQ ID Nos: 23, 25, 27, 29, 31, 33, 35, 37); or (b) a DNA sequence encoding a
Haemophilus influenzae
adhesin (Hia) protein having an amino acid sequence selected from the group consisting of those shown in
FIGS. 18
,
19
,
20
,
21
,
22
,
23
,
24
and
25
(SEQ ID Nos: 24, 26, 28, 30, 32, 34, 36, 38).
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode the Hia protein from strain 11 or 33 of non-typeable Haemophilus.
In another aspect of the present invention, there is provided an isolated and purified nucleic acid molecule encoding an N-truncated
Haemophilus influenzae
adhesin (Hia) protein of a strain of
Haemophilus influenzae
which is amplifiable by a pair of nucleotides which are selected from the group consisting of SEQ ID No: 7 and SEQ ID No: 15; SEQ ID No: 9 and SEQ ID No: 15; SEQ ID No: 11 and SEQ ID No: 15; SEQ ID No: 13 and SEQ ID No: 15.
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode an N-truncated Hia protein from strain 11 or 33 of non-typeable Haemophilus, starting at codon V38.
The plasmid vector incorporating the isolated and purified nucleic acid provided in accordance with these aspects of the invention may have the identifying characteristics of a plasmid which is selected from the group consisting of:
DS-2008-2-3 as shown in
FIG. 1A
DS-2186-1-1 as shown in
FIG. 5A
DS-2201-1 as shown in
FIG. 5A
DS-2186-2-1 as shown in
FIG. 5A
DS-2168-2-6 as shown in
FIG. 5A
The vector provided herein may include the cer gene from
E. coli
. Accordingly, in another aspect of the present invention, there is provided a vector for transforming a host, comprising a nucleic acid molecule encoding a full-length or N-truncated
Haemophilus influenzae
adhesin (Hia) protein, a promoter for expression of said full-length or truncated Hia protein and, optionally, the cer gene of
E. coli
. The vector may be a plasmid vector or other non-replicating vector, which may have the identifying characteristics of a plasmid vector which is selected from the group consisting of:
BK-96-2-11 as shown in
FIG. 6A
DS-2242-1 as shown in
FIG. 7A
DS-2242-2 as shown in
FIG. 7A
DS-2340-2-3 as shown

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