Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-08
2002-08-13
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069300, C435S069700, C435S071100, C435S243000, C435S252300, C435S320100, C536S023100, C536S023700, C536S024100, C424S256100
Reexamination Certificate
active
06432669
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular genetics and, in particular, to the production of recombinant
Haemophilus influenzae
high molecular weight proteins and nucleic acid molecules and vectors employed therein.
BACKGROUND TO THE INVENTION
Encapsulated
Haemophilus influenzae
type b strains are a major cause of bacterial meningitis and other invasive infections in young children. However, the non-encapsulated or nontypeable
H. influenzae
(NTHi) are responsible for a wide range of human diseases, including otitis media, epiglottitis, pneumonia and tracheobronchitis. Vaccines based upon
H. influenzae
type b capsular polysaccharide conjugated to diphtheria toxoid (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), tetanus toxoid (ref. 2 and U.S. Pat. No. 4,496,538), or
Neisseria meningitidis
outer membrane protein (ref. 3) have been effective in reducing
H. influenzae
type b-induced meningitis, but not NTHi-induced disease (ref. 4).
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. 5). 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. Barenkamp and Bodor (ref. 6) demonstrated that convalescent sera from children suffering from otitis media due to NTHi contained antibodies to high molecular weight (HMW) proteins. About 70 to 75% of NTHi strains express the HMW proteins and most of these strains contain two gene clusters termed hmw1ABC and hmw2ABC. The hmwA genes encode the structural HMWA proteins and the hmwb and hmwC genes are accessory genes responsible for the processing and secretion of the HMWA proteins (refs. 7, 8, 9; U.S. Pat. No. 5,603,938; Wo 97/36914). The HMWA proteins have been demonstrated to be adhesins mediating attachment to human epithelial cells (ref. 10) and only properly processed HMWA proteins appear to be effective adhesins (ref. 8). Immunization with a mixture of native HMW1A and HMW2A proteins resulted in protection in the chinchilla intrabulla challenge model of otitis media (ref. 11; WO 97/36914). The prototype hmw1A gene from NTHi strain 12 encodes a 160 kDa HMW1A protein that is processed by cleavage of a 35 kDa amino terminal fragment, generating the mature 125 kDa HMW1A protein. Similarly, the NTHi strain 12 hmw2A gene encodes a 155 kDa HMW2A protein that is processed by cleavage of a nearly identical 35 kDa amino terminal fragment to produce the mature 120 kDa HMW2A protein.
Plasmid pHMW1-15 (ref. 8) has a pT7-7 backbone (ref. 12) and contains the complete NTHi strain 12 hmw1ABC operon with 5′- and 3′-flanking regions. There are about 400 bp of 5′-flanking sequences located between the T7 promoter and the start of the hmw1A structural gene. Plasmid pHMW2-21 (ref. 10) has a pT7-7 backbone and contains the complete hmw2ABC operon with 5′- and 3′-flanking sequences. There are about 800 bp of 5′-flanking sequences located between the T7 promoter and the start of the hmw2A structural gene. The rHMW1A and rHMW2A proteins are produced in relatively low yield from plasmids pHMW1-15 and pHMW2-21.
The
H. influenzae
hmw1 ABC or hmw2 ABC genes can be genetically engineered to produce the mature recombinant HMW1A or HMW2A proteins by deleting the sequence encoding the 35 kDa leader sequence, that is normally removed by processing in
H. influenzae
. Since the leader sequence has been deleted, there should be no necessity for the hmw1BC or hmw2BC genes which serve to process and secrete the mature HMW1A and HMW2A structural proteins in
H. influenzae
(ref. 9). The yield of rHMW1A or rHMW2A protein can be significantly increased by deletion of the leader sequence and processing genes, however, the purified recombinant proteins are not protective. As set forth herein, the hmw1BC and hmw2BC genes or their protein products apparently contribute to the protective ability of rHMW1A and rHMW2A proteins. Such a requirement for otherwise redundant accessory genes, is unexpected.
The
E. coli
cer gene is thought to stabilize plasmids by preventing multimerization (ref. 13). For expression vectors with large inserts, the cer gene may be used to stabilize the plasmids.
SUMMARY OF THE INVENTION
The present invention is directed towards the provision of recombinant non-typeable
H. influenzae
high molecular weight proteins that are protective by providing certain nucleic acid molecules and vectors containing the same.
It has now been found that, in order to obtain recombinant high molecular weight (HMW) proteins of non-typeable Haemophilus which are protective, it is necessary to provide a vector containing only the segment of the A portion of the operon which encodes the mature HMW protein, i.e. lacking the segment of the A gene which encodes the leader sequence, and the B and C portions of the operon. It has also been found that the level of expression of the mature protein may be enhanced by including in the vector at least one additional segment which encodes the mature protein, the cer gene from
E. coli
or both. Accordingly, in one aspect of the present invention, there is provided a nucleic acid molecule comprising a promoter functional in
E. coli
and operatively coupled to a modified operon of a non-typeable strain of Haemophilus comprising A, B and C genes, wherein the A gene of the operon contains only a nucleic acid sequence which encodes a mature high molecular weight protein of the non-typeable strain of Haemophilus, and hence from which the portion of the A gene encoding the leader sequence is absent.
Any suitable promoter may be used to effect expression of the mature HMW protein in
E. coli
. However, it is preferred to use the T7 promoter.
The encoded mature high molecular weight protein may be HMW1 or HMW2 protein of the non-typeable Haemophilus strain. The non-typeable Haemophilus strain may be selected from the group consisting of strains 12, Joyc, K21, LCDC2, PMH1 and 15 of non-typeable
Haemophilus influenzae.
The present invention also provides the nucleotide sequences for the hmw1A and/or hmw2A genes of certain non-typeable strains of
Haemophilus influenzae
which have not been previously isolated, purified and expressed, along with the deduced amino acid sequences of the corresponding HMW1 and HMW2 proteins of the non-typeable Haemophilus strains.
Accordingly, in another aspect of the invention, there is provided an isolated and purified nucleic acid molecule encoding a high molecular weight (HMW) protein of a non-typeable 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
,
25
,
26
and
27
(SEQ ID NOS: 25, 27, 29, 32, 33, 36, 38, 40, 42, 44, 46, 48, 50, 52,
Klein Michel H.
Loosmore Sheena M.
Yang Yan-Ping
Aventis Pasteur Limited
Graser Jennifer E.
Sim & McBurney
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