Title of Invention

POLYPEPTIDES AND POLYNUCLEOTIDES FROM MORAXELLA CATARRHALIS

Abstract The invention provides BASB027 polypeptides and polynucleotides encoding BASB027 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.
Full Text BASBO

21 PROTEINS AND GENES FROM MORAXELLA CATARHALIS, ANTIGENS. ANTIBODIES. AND USES

FIELD OF THE INVENTION
This invention relates to polynucleotides, (herein rererred to as "BASBOI" polynucieotidets)") polypeptides encoded by them (referred to herein as "BASBOZ'" or " BASBGl" polypeptidets)") recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such poiypepiides and polynucleotides, including vaccines against bacterial infections. In a further aspect. The invention relates to diagnostic assays for detecting infection of certain pathogens.
BACKGROUND OF THE INVENTION
Moraxella cararrhalis ('also named Branhameila catarrhalis is a Gram negative bacteria frequently isolated from the human upper respirator.' tract. It is responsible for several pathologies the main ones being otitis media in infants and children, and pnemonia in elderiies. It is also responsible of sinusitis, nosocomial infections and less frequently of invasive diseases.
Otitis media is an important childhood disease both by the number of cases and its potential sequelae. More than 3.5 millions cases are recorded every year in the United States, and it is estimated that 80 % of the children have expenenced at least one episode of otitis before reaching the age of 3 (Klein. JO (1994) Clm.Inf.Dis 19:823). Left untreated, or becoming chronic, this disease may lead to hearing losses that could be temporary (in the case of fluid accumulation in the middle ear) or permanent (if the auditive nerve is damaged). In infants. such hearing losses may be responsible for a delayed speech learning.
Three bacterial species are primarily isolated from the middle ear of children with otitis media: Streptococcus pneumoniae, non typeable Haemophilus influoiza ("NTHi and M cararrhalis. They are present in 60 to 90 % of the cases. A review of recent studies shows
that S.Pneumoniae and NTHi represent both about 30% and M. catarrihalis about 15% of


KL et al. (1990) .Am.J.Med. 88 (suppl. 5A):28S). Serum resistance could therfore be


SUMMARY OF THE INVNTION




preferably at least 95% identity, most preferably at least 97-99% identity. to that of SEQ ID NO:2 or 4 over the ensure length of SEQ ID NO::


preferably at leasr 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 or 4 over the entire length of SEQ ID NO:2

Afragment is a polypeptide havin an amino acid sequence that is entirely the same as part bur not all of any amino acid sequence of any polypeptide of the invention. As with BASBCZ polypeptides, fragments may be "free-standing," or comprising within a larger polypeptide of which they from a part or region, most preferably as a single continuous region m a single larger polypeptide.

Fragments of the polypepddes of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis: therefore, these fragments may be employed as intermediates for producing the full-length polypeptides of the invention.


The proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protem. The fusion panner may assist in providing T helper epitopes


Polypeptides of the present invention can be prepared in any suitable maimer. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a




Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene


(h) a polynucleotide sequence encoding a polypeptide which has at least 85% identity prererably at least 90oC identity. more preferably at least 95% identity. Even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ IDNO:2 or 4. over the entire length of SEQ ID NO:2 or 4 respectively.
A polynucleotide encoding a polypeptide of the present invention, including homologs and onhologs from species other than Moraxella calarrhalis. may be obtained by a process which comprises the steps of screening an appropriate libran' under stringent hybridization


identical to the polypeptide encoding sequence contained in nucleoudes 1 to 2439 of SEQ ID NO: 1 or 3 respective!}. Alternatively it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID


Further preferred embodiments of the invention are polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB027 polypeptide having an amino acid sequence set out in SEQ IDNO:2 or 4. and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 90% identical over its entire length to


(1989). particularly Chapter ! 1 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.


There are several methods available and well known to those skilled in the art to obtain full-length DNAS. or extend short DNAs. for example those based on the method of Rapid Amplification of cDNA ends (R-ACE) (see, for example. Frohman. et al. PSAS USA S5: 8998-9002, 1988). Recent modifications of the technique, exemplilled by the MarathonTM technology) ('Clontech Laboratories Inc.) for example, have significantly simplified the


The in\'ennon also provides polynucleoiidcs that encode a polypeptide that is the matin-e protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As


In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in
particular genetic immunization.


Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that compose a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques.
For recombmant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or ponions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be


A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids. from bacteriophage, from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picomaviruses, retroviruses. , and aiphaviruses and vectors denved from combinations thereof, such as those derived from piasmid and bactenophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any




out with or without denaturing agents. Polynucleotide differences may also be detected by direct DN'A or RNA sequencing See. for example. Myers et al. Science, 230: 1242 (1985).


out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing See. for example, Myers et al. Science. 230: 1242 (1985).


preferably SEQ IDNO:1 or 3, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a suscentibility to a disease.


increased level of expression of polynucleotide having a sequence of SEQ IDNO:1 or 3. increased or decreased expression of a BASB027 polynucleotide can be measured using any on of the methods well known in the an for the quantitation of polynucleotides, such


The polynucleotides of the invention may be used as components of polynucleotide arrays. preferably- high density arrays or grids. These high density arrays are panicuiarly useful for diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing- such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual. Such a presence may indicate the presence of a pathogen, panicuiarly Moraxclla catarrhalis. and may be useful in diagnosing and/or prognosing disease or a course of disease. A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO: 1 or 3 are preferred. Also preferred is a comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2 or 4.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.


Techniques for the production of single chain antibodies (U.S. Patent No. 4.946.778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms or animals, such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.


The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by. for exampie. affinity chromatography.

Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For exampie. if the individual is human the antibody may most preferably be "humanized." where the complimentarity determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. ('1986). Nature 321. 522-525 or Tempest et al., (1991) Biotechnology- 9, 266-273.
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in. for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See. e.g.. Coligan et al.



The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/'or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the an. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.


.Another example of an assay for BASB027 agonists is a competitive assay that combines BASB027 and a potential agonist with BASB027-binding molecules, recombinant BASB027 binding molecules, natural substrates or ligands. or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. BASB027 can be labeled, such as by radioactivity or a colorimetric compound, such that the number of BASB027 molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.


incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such


In accordance with yet another aspect of the invention, there are provided BASB027 agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.


Alternatively, peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to ami-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
Vaccines




Such experiments will be panicularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, panicularly Moraxella catarrhalis infection, in mammals, particular humans.


An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses ('humoral response).
Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. TH2-

lype immune responses are characterised by the generation of a broad range of immunogiobuiin isorypes including in mice IgGL IgA. and IgM.

It is known thai cenain vaccine adjuvants are panicularly suited to the stimulation of either TH1 or TH2 - type cytokine responses. Traditionally the best indicators of the TH1 :TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgGl:IgG2a ratio of antigen specific antibody responses.


Non-reactogenic adjuvant formulations containing QS21 ave been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THl stimulating adjuvants when formulated together with an antigen.


Preferably a carrier is also present in the vaccine composition according to the invention. The carrier mav be an oil in water emulsion, or an aluminium sail, such as aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene. alpha tocopherol and Tween 80. In a panicuiariy preferred aspect the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 and'or lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of lug - 200µg. such as 10-100µg, preferably 10µg - 50µg per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of l%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.


Compositions, kits and administration
In a further aspect of the invention there are provided compositions comprising a BASB027 polynucleotide and or a BASB027 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/'or a polypeptides discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical
J-

carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypepnde and or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to. saline, buffered saline, dextrose, water, glyverol ethanol and combinations thereof The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypepnde and/or polynucleotide, such as the soluble form of a polypeptide and/'or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal. can be used. Altemative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and'/or localized, in the form of salves, pastes, gels, solutions. powders and the like.
For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg. typically around 1 mg kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course,
be individual instances where higher or lower dosage ranges are merited, and such are

within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 µg/kg of subject.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an

interval of 1-3 weeks. With the indicated dose range, no adverse toxicoiogical etYccts will be obser\'ed with the compounds of the invention which would preclude their administration to suitable individuals.

Also provided by the invention are methods for the analysis of character sequences or strings, panicularly genetic sequences or encoded protein sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis. DNA. UNA and protein structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, nucleic acid base trimming, and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification. This method comprises the steps of: providing a first polynucleotide sequence comprising the

sequence of a polynucleotide of the invention in a computer readable median:; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
A computer based method is also provided for performing homology idetification said method comprising the steps of; providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify' homology.
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.
DEFINITIONS
"Identity," as known in the art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in {Computational Molecular Biology, Lesk. .A.M.. ed.. Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DAV., ed.. Academic Press, New York, 1993; Computer Analysis of Sequence Data, Pan I, Griftln, A.M.. and Griffin, H.G., eds., Humana Press. New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine,

G.. Academic Press. 19S7; and Seauence Analysis Primer, Gribskov. M. and Devereux, J. eds.. M Stockton Press. New York. 1991: and Carillo, H.. and Lipman. D.. SIAM. Applied Math.. -48 1073 (] 988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity- are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to. the GAP program in the GCG program package (Devereux. J., et al., Xucleic Acids Research I2d 387 (1984)). BLASTP. BLASTN (Alt.schul. S.F. et al.. J. Molec. Biol 215: 403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul. S., etui, NCBI NLM NIH Bcthesda, MD 20894; Altschul. S.. et al.J. Mul. Biol. 2/:: 403-410 (1990). The well known Smith Waterman algorithm mav also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap" program from
Genetics Computer Group. Madison W1. The aforementioned parameters are the default
parameters for pepdde comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group. Madison WI. These arc the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be. are provided m (I) and (2) below.
(1) Polynucieotide embodiments funher include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60. 70. 80, 85, 90, 95. 97 or 100% identity to the reference sequence of SEQ IDNO:1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ IDNO:1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleodde deletion, substitution, including transition and transversion. or insenion. and wherein said alterations may occur at the 5" or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal posidons. interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucieondes in SEQ IDNO:1 by the integer defining the percent idennty divided by 100 and then subtracting that product from said total number of nucleotides in SEQ IDNO:1, or:
wherein xn is the number of nucleodde alterations, Xn is the total number of nucleotides in SEQ ID NO: 1. y is 0.50 for 50%. 0.60 for 60%, 0.70 for 70%, 0.80 for 80%. 0.85 for
85%, 0.90 for 90%. 0.95 for 95%. 0.97 for 97% or 1.00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from Xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ IDNO:2 may create nonsense, missense or



said alterations are selected from the group consisting of at least one amino acid deletion, substitution, includmg conserrvative and non-conservative substitution, or insenion. and wherein said alterations may- occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ IDNO;2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ IDNO:2. or:
na wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO::, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%. 0.85 for
85°/o, 0.90 for 90%. 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ IDNO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insenion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino

acids in SEQ IDNO:2 by the integer denning the percent identity divided by- 100 and then subtracting that product from said total number of amino acids in SEQ IDNO:2. or:
na wherein na is the number of amino acid alterations, xa is the total number or" amino acids in SEQ ID NO:2. y is. for instance 0.70 for 70°o. 0,80 for 80%. 0.85 for,85% etc.. and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
"Individual(s)." when used herein with reference to an organism, means a multicellular eukaryote. mciuding. but not limited to a metazoan. a mammal, an ovid. a bovid. a simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e.. if it occurs in namre. it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DN.A or modified RNA or DNA including single and double-stranded regions.
'Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a

polynucleotide differs in nucleolide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences arc limited so that the sequences of the reference polypeptide and the variant are closely similar overall and. in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or insened amino acid residue mav or mav not be one encoded bv the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or bv direct svnthesis.
"Disease(s)" means any disease caused by or related to infection by a bacteria, including, for example, otitis media in infants and children, pneumonia in elderlies, sinusitis, nosocomial infections and invasive diseases, chronic otitis media with hearing loss, fluid accumulation in the middle ear. auditive nerve damage, delayed speech learning, infection of the upper respiratory tract and inflammation of the middle ear.

EXAMPLES:
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the an, except where otherwise described in detail. The examples are illusiran\e. but do not limit the invention.
Example 1: ,
Discovery and confirmaton DNA sequencing of the BASB027 gene from Moraxella catarrlialls strain AT CC 43617,
The BASB027 gene of SEQ IDNO:1 was first discovered in the Incyte PathoSeq data base containing unfinished genomic DNA sequences of the Moraxella catarrhalis strain ATCC 4367 (also referred to as strain Mc2931). The translation of the BASB02" polynucleotide sequence. shown in SEQ IDNO:2, showed significant similarity (32 % identity in a 817 amino acids overlap) to the OMP85 outer membrane protein of Neisseria meningitidis.
The sequence of the BASB027 gene was funher confirmed experimentally. For this purpose, genomic DNA was extracted from 1010cells of the M. catarrhalis cells (strain ATCC 43617) using the QIAGEN genomic DNA Extraction kit (Qiagen Gmbh), and lug of this material was submitted to Polymerase Chain Reaction DNA amplification using primers E5I5515 (5'- ACT-ATA-GGG-CAC-GCG-TG -3') [SEQ IDNO:5] and E5I552S : (5'- CCT-GCG-TTT-GTT-TGA-TTG-AG-3') [SEQ IDNO:6]. This PCR product was purified on a Biorobot 9600 (Qiagen Gmbh) apparatus and subjected to DNA sequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and an ABI 377/PRISM DNA sequencer. DNA sequencing was performed on both strands with a redundancy of 2 and the full length sequence was assembled using the SeqMan program from the DNASTAR Lasergene software package. The resulting DNA sequence and deduced polypeptide sequence are shown as SEQ IDNO:3 and SEQ IDNO:4


Example 2:
\'ariability analysis of the BASB027 gene among several Moraxella cararrhalis
strains.
2A: Restriction Fragment Length Analysis (RFLP).
Genomic DNA was extracted from 16 M catarrhalis strains (presented in Table 1) as described below. M, catarrhalis was streaked for single colonies on BHI agar plates and grown overnight at 37 °C. Three or four single colonies were picked and used to inoculate a -1.5 ml BHI (Brain-hean infusion) broth seed culture which was grown overnight in a shaking incubator. -300 rpm, at 37 °C. A 500ml erlenmeyer flask containing -150 mi of BHI broth was inoculated with the seed culture and grown for -12-16 hours at 37 °C in a shaking incubator, -175 rpm, to generate cell mass for DNA isolation. Ceils were collected by centrifugation in a Sorvall GSA rotor at -2000 X g for 15 minutes at room temperature. The supernatant was removed and the cell pellet suspended in -5.0 ml of sterile water. An equal volume of lysis buffer (200 mM NaCl, 20 mM EDTA. 40 mM Tris-Hcl. pH 8.0, 0.5% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol and 250 µg/ml of proteinase K) was added and the cells suspended by gentle agitation and trituration. The cell suspension was then incubated -12 hours at 50oC to lyse the bacteria and liberate chromosomal DNA. Proteinaceous material was precipitated by the addition of 5.0 ml of saturated NaCl (-6.0 M, in sterile water) and centrifugation at -5.500xg in a Sorvall SS34 rotor at room temperature. Chromosomal


products were separated by agarose or polyacrylamide gel electrophoresis using standard molecular biology procedures as described in 'Molecular Cloning, a Laboratory Manual. Second Edinon. Eds: Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989". The photographs of the resulting electrophoresis gels are displayed in Figure 1. For each strain, RFLP patterns corresponding to the 6 restriction enzymes were scored and combined. Groups of strains sharing identical combination of RFLP patterns were then defined. Using this methodology, the strains tested in this study fell into 4 genomic groups (Group 1: Mc2906, Mc 2908. Mc29I2, Mc2926; Group 2: Mc2905. Mc2907, Mc2909, Mc2911, Mc2913, Mc2960, Mc2975 ; Group 3: Mc2910. Mc2912, Mc2956. Mc2969; Group 4: Mc293I). These data suppon that the Moraxella catarrhalis population used in this study displays limited nucleotide sequence diversity for the BASB027 gene.

Table 1: Features of the Moraxella catarrhallis strains used in this study


Example 3: Construction of Plasmid to Express Recombinant BASB027
A: Cloning of BASBO:".
The BamHl and Sad restriciion sites engineered into the forward ([SEQ ID NO:"']) and reverse complementary ([SEQ ID NO;8]) amplification primers, respectively, permitted directional cloning of an -2500 bp PCR product into the commercially available E.coli expression plasmid pQE3.) (QiaGen. ampicillin resistant) such that a mature BASB02" protein could be expressed as a fusion protein containing a ('His)6 affinity-chromatography tag at the N-terminus. The BASB027 PCR product was purified from the amplification reaction using silica gel-based spin columns (QiaGen) according to the manufacturers instructions. To produce the required Bamlil and Sail termini necessary for cloning, purified PCR product was sequentially digested to completion with BamHl and Sail restriction enzymes as recommended by the manufacturer (Life Technologies). Following the first restriction digestion, the PCR product was purified via spin column as above to remove salts and eluted in sterile water prior to the second enzyme digestion. The digested DNA fragment was again purified using silica gel-based spin columns prior to ligation with the pQE30 plasmid.
B: Production of Expression Vector.
To prepare the expression piasmid pQE30 for ligation, it was similarly digested to completion with both BamHl and Sad and then treated with calf intestinal phosphatase (CIP, -0.02 units / pmole of 5' end. Life Technologies) as directed by the manufacturer to prevent self ligation. An approximately 5-fold molar excess of the digested fragment to the prepared vector was used to program the ligation reaction. A standard -20 µl ligation reaction (-16°C, -16 hours), using methods well known in the an, was performed using T4 DNA ligase (-2.0 units / reaction. Life Technologies). .An aliquot of the ligation (-5 ul) was used to transform electro-competent M15(pREP4) cells according to methods well known in the art. Following a -2-3 hour outgrowth period at 37°C in -1.0 ml of LB broth, transformed cells were plated on LB agar plates

containing kanamycin (50 µg/ml) and ampiciliin (100 µg/ml). Both anribioties were included in the selection media to ensure that all transformed cells carried both the pREP4 plasmid (KnR), which carries the laclq gene necessary for the repression of expression for IPTG-inducible expression of proteins on pQE30. and the pQE30-BASB02" piasmid (ApR). Plates were incubated overnight at 37°C for -16 hours. lndividual IvnR ApR colonies were picked with sterile toothpicks and used to "patch" inoculate fresh LB KNR . ApR plates as well as a --1.0 ml LB KnR ' ApR broth culture. Both the patch plates and the broth culture were incubated overnight at 3^-C in either a standard incubator (plates) or a shaking water bath.
A whole cell-based PCR analysis was employed to verify that transformants contained the BASBOZ" DNA insert. Here, the -1.0 ml overnight LB Kn / Ap broth culture was transterred to a 1.5 ml polypropylene tube and the cells collected by centrifugation in a Beckmann microcentrifuge (-3 min.. room temperature. -12,000 X g). The cell pellet was suspended in -200µl of sterile water and a -10µl aliquot used to program a ~-50µl final volume PCR reaction containing both BASB027 forward and reverse amplification primers. Final concentrations of the PCR reaction components were essennally the same as those specified in example 2 except -5.0 units of Tag polymerase was used. The initial 95'C denaturation step was increased to 3 minutes to ensure thermal disruption of the bacterial cells and liberation of plasmid DNA. An ABI Model 9700 thermal cycler and a 32 cycle, three-step thermal amplification profile, i.e. 95oC. 45sec; 55-58oC. 45sec. 72°C, 1min.. were used to amplify the BASB027 PCR fragment from the lysed transformant samples. Following thermal amplification, a -20^1 aliquot of the reaction was analyzed by agarose gel electrophoresis (0.8 % agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by UV illumination after gel electrophoresis and ethidium bromide staining. A DNA molecular size standard (1 Kb ladder. Life Technologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the PCR products. Transformants that produced the expected - 2500 bp PCR product were identified as strains containing a BASB027




Fluorescent dye-labelled termination products were purified using microcentrifuge size-exclusion chromatography columns (Princeton Genetics), dried under vacuum, suspended in a Template Resuspension Buffer (Perkin-Elmer) for capillary electrophoresis or deionized formamide for PAGE, denatured at 95°C for -5 min, and analyzed by high resolution capillar)' electrophoresis (ABI 310 Automated DNA Sequenator. Perkin-Elmer) or high resolution PAGE (ABI 577 Automated DNA




Aminoethyl)-benzenesulfonylfuoride), Complete protease inhibitor cocktail tablets, and PMSF (phenyimethyl-sulfonylfluoride) were obtained from Roche Diagnostics Corporation, Indianapolis. Indiana. Bestatin, Pepstatin A, and E-64 protease inhibitor were obtained from Calbiochem, LaJolla, California. Dulbecco's Phosphate Buffered Saline(lx PBS) was obtained from Quality Biological, Inc., Gaithersburg, Maryland. Dulbecco's Phosphate Buffered Saline (10x PBS) was obtained from BioWhitlaker,


Cell paste was thawed at room temperature for 30 t6 60 minutes. Five to six grams of material was weighed out into a 50 ml disposable centrifuge tube. To this five3 mis/gram of Guanidine hydrochloride (Gu-HCl) buffer was added (6 M Guanidine hydrochloride. 100 mM Sodium phosphate, monobasic, 10 mM Tris and 0.05 % Triton X-100. pH 8.0). Cell paste was resuspended using a PRO300D proscientific homogenizer. at 3-/4 power for one minute. The extraction mixture was then placed at room temperature with gentle agitation for 60 to 90 minutes. After 60 to 90 minutes the extraction mixture was centrifuged at 15,800 x g for 15 minutes (Sorvall RC5C centrifuge. 11.500 rpm). The supernatant (SI) was decanted and saved for additional purification. The pellet (PI) was saved for analysis.




Protein Sequencing
— — -■ • •—
Amino terminal amino acid sequencing of the purified protein was performed to confirm the production of the correct recombinant protein using well defined chemical protocols on Hewlett-Packard model G1000A sequencer with a model 1090 LC and a Hewlett-Packard model 241 sequencer with a model 1100 LC.
Example 5 : Production of Antisera to Recombinant BASB027
Polyvalent antisera directed against the BASB027 protein were generated by vaccinating two rabbits with the purified recombinant BASB027 protein. Each animal is given a total of three immunizations intramuscullarly (i.m.) of about 20µg BASB027 protein per injection (beginning with complete Freund's adjuvant and followed with incomplete Freund's adjuvant) at approximately 21 day intervals. Animals were bled prior to the first immunization ("pre-bleed") and on days 35 and 57.
Anti-BASB027 protein titres were measured by an ELISA using purified recombinant BASB027 protein (0.5 ug/'well). The titre is defined as the highest dilution equal to or


(360 mM Tris buffer, pH 8.8. containing 4% sodium dodecylsulfate and 20% glycerol), and incubating the suspension at 100°C for 5 minutes. The solubilized cells were resolved on 4-20% polyacrylamide gels and the separated proteins were electrophoretically transferred to PVDF membranes at 100V for 1 hour as previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5 % non-fat dry milk. All subsequent incubations were carried out using this pretreatment buffer
PVDF membranes were incubated with 25ml of a 1:500 dilution of preimmune serum or rabbit immune serum for Ihour at room temperature. PVDF membranes were then washed twice with wash buffer (20 mM Tris buffer, pH 7.5, containing 150 mM sodium chloride and 0.05% Tween-20). PVDF membranes were incubated with 25ml of a 1:5000 dilution of peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories. West Grove, PA) for 30 minutes at room temperature. PVDF membranes were then washed 4 times with wash buffer, and were developed with 3-amino-9-


Complement-mediated cytotoxic activity of anti-BASB027 antibodies was examined to determine the vaccine potential of BASB027 polypeptide. Antiserum was prepared as described above. The activities of the pre-immune serum and the anti- BASB027 antiserum in mediating complement killing of M. catarrhalis were examined using the "Serum Bactericidal Test" described by Zollinger et al. (Immune Responses to Neissaeria
meningitis, in Manual of Clinical Laboratory Immunology, 3rd ed., pg 347-349). except that cells of A/ catarrhalis strains or cultivars were used instead of Neisseria meningiris cells.
The bactericidal titer of rabbit antiserum (50% killing of homologous strain) was 1:128 (immune).
Example 7 : Presence of Antibody to BASB027 in Human Convalescent Sera
Western blot analysis of purified recombinant BASB027 were performed as described in Example 4 and 6 above, except that a pool of human sera from children infected by A/, caiarrhaiis was used as the first antibody preparation. Results show that antisera from naturally infected individuals react to the purified recombinant.
Example 8 : Production of BASB027 peptides. Antisera and Reactivity Thereof

Two slon amino acid BASB0Z7 specific peptides, having the sequences oF CYAKPLNKKQNDQTDT (SEQ IDNO:9) and YLTARRGQQTTLGEVVC (SEQ ID
NO: 10) were produced in the laboratory using generally well known method?. These peptides coupled to KLH were used to produce antibodies in 12 weeks old Spcciric Pathogen Free New-Zealand female rabbits. Rabbits received 4 injections ar approximately 3 weeks intervals of 200 µg of peptide-KLH in complete ( 1 injectin or incomplete (2' and 4" injections) Freund's adjuvant. Animals were bled prior to the first immunization and one month after the 4'" injection.
Anti-peptide mid-point litres were measured by an ELISA using free peptides. Anti-peptide Mid-point titres one month after the 4'" immunization were superior to 15000. Western blots of purified recombinant BASB027, using anti-peptide antibodies as the first antibody, were prepared as described in Example 4 and 6. The results are presented m Figure 8.


assigned deposit number 207105.
The sequence of the polynucleotides contained in the deposited strain / clone, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
The deposit of the deposited strains have been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required for enablement, such as that required under35 U.S.C. §112.

















SEQUENCE INFORMATION
BASB027 Polynucleotide and Polypeptide Sequences








WE CLAIMS:
1. An isolated polypeptide comprising an amino acid sequence which has at least 85% identity to the amino acid sequence seieaed from the group consisting of: SEQ IE' NO;2 and SEQ ID NO:4, over the entire length of SEQ ID NO:2 or SEQ ID NO:4 respe.rively.
2. An isolated polypeptide as claimed in claim 1 in which the amino acid sequence has at leas: 95% identity to the amino acid sequence selected from the group consisting of: SEQ ID NO:2 and SEQ ID NO:4, over the entire length of SEQ ID NO:2 or SEQ ID NO:4 respectively,
3. The polypeptide as claimed in claim 1 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO:2 and SEQ ID NO:4.
4. An isolated polypeptide having the amino acid sequence selected from the group consisting of SEQ ID No:2 or SEQ ID NO:4.
5. An immunogenic fragment of the polypeptide as claimed in any one of claims 1 to 4 in which the immunogenic fragment is capable of raising an immune response (if necessary when coupled to a carrier) which recognises the polypeptide of SEQ ID NO:2 or SEQ ID NO:4.
6. A polypeptide as claimed in any of claims 1 to 5 wherein said polypeptide is pan of a larger fusion protein.
7. An isolated polynucleotide encoding a polypeptide as claimed in any of claims 1 to 6.
8. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has ax least 85% identity to the amino acid sequence of SEQ ID NO:2 or 4 over the entire length of SEQ ID NO:2 or 4 respectively; or a nucleotide sequence complementary to said isolated polynucleotide.

9. An isolated polynucleotide comprising a nucleotide sequence that has at least 85% identity
to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2 or 4 over the entire coding
region; or a nucleotide sequence complementary to said isolated polynucleotide.
10. An isolated polynucleotide which comprises a nucleotide sequence which has at least 85% identity to that of SEQ ID NO: I or 3 over the entire length of SEQ ID NQ: 1 or 3 respectively; or a nucleotide sequence complementary to said isolated polynucleotide.
11. The isolated polynucleotide as claimed in any one of claims 7 to 10 in which the identity is at least 95% to SEQ ID NO: 1 or 3.
12. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2 or SEQ ID NO:4.
13. An isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or SEQ ID NO:3,
14. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2, SEQ ID NO:4 obtainable by screening an appropriate library under stringent hybridization condhions with a labeled probe having the sequence of SEQ ID NO: 1 or SEQ ID NO;3 or a fragment thereof

15. An expression vector or a live microorganism comprising an isolated recombinant polynucleotide according to any one of claims 7 -14.
16. A host ceil comprising the expression vector of claim 15 expressing an isolated polypeptide comprising an amino acid sequence that has at least 85% identity to the amino acid sequence selected from the group consisting of: SEQ ID NO:2 and SEQ ID NO:4, or a membrane of the host cell comprising the expressed polypeptide.


17. A process for producing a polypeptide of claims 1 to 6 comprising culruring a host cell of claim 16 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium,
18. A process for expressing a polynucleotide of any one of claims 7-14 comprising transforming a host cell with the expression vector comprising at least one of said polynucleotides and culturing said host cell under conditions sufficient for expresi:ion of any one of said polynucleotides.
19. A vaccine composition comprising an effeciive amount of the polypeptide of any one of claims I to 6 and a pharmaceutically acceptable carrier.
20. A vaccine composition comprising an effective amount of the polynucleotide of any one of claims 7 to 14 and a pharmaceutically effective carrier.
21. The vaccine composition according to either one of claims 19 or 20 wherein %iid composition comprises ax least one other Neisseria meningindis antigen.
22. An antibody immunospecific for the polypeptide or immunological fragment as claimed in any one of claims 1 to 6.
23. A method of diagnosing a Neisseria meningitidis infection, comprising identifying a polypeptide as claimed in any one of claims I - 6, or an antibody that is immunospecific for said polypeptide, present within a biological sample from an animal suspected of having such an infection,
24. Use of a composition comprising an immunologically effective amount of a polypeptide as claimed in any one of claims 1 - 6 in the preparation of a medicament for use in gcneranng an immune response m an animal.

25. use Ota composition comprising an immunologically effective amouut of a polyaucleotide as claimed in any one of claims 7 -14 in the prcparation of a medicament for use in generating an immune response in an animal.
26. A therapeutic composition useful in treating humans with Neisseria meningitidis comprising at least one antibody directed against the polypeptide of claims 1 - 6 and a suitable pharmaceutical carrier.
27. An isolated polypeptide substantially as herein described, with reference to the accompanying drawings.
28. A vaccine composition, substantially as herein described, with reference to the accompanying drawings.
29. A therapeutic composition, substantially as herein described, with reference to the accompanying drawings.


Documents:

IN-PCT-2000-751-CHE AMANDED CLAIMS 11-02-2010.pdf

IN-PCT-2000-751-CHE AMANDED PAGES OF SPECIFICATION 11-02-2010.pdf

IN-PCT-2000-751-CHE CORRESPONDENCE OTHERS 11-02-2010.pdf

IN-PCT-2000-751-CHE FORM-1 11-02-2010.pdf

IN-PCT-2000-751-CHE FORM-3 11-02-2010.pdf

IN-PCT-2000-751-CHE OTHER PATENT DOCUMENT 11-02-2010.pdf

in-pct-2000-751-che-claims.pdf

in-pct-2000-751-che-correspondence others.pdf

in-pct-2000-751-che-correspondence po.pdf

in-pct-2000-751-che-description complete.pdf

in-pct-2000-751-che-drawings.pdf

in-pct-2000-751-che-form 1.pdf

in-pct-2000-751-che-form 19.pdf

in-pct-2000-751-che-form 26.pdf

in-pct-2000-751-che-form 3.pdf

in-pct-2000-751-che-form 5.pdf

in-pct-2000-751-che-pct.pdf


Patent Number 239261
Indian Patent Application Number IN/PCT/2000/751/CHE
PG Journal Number 12/2010
Publication Date 19-Mar-2010
Grant Date 15-Mar-2010
Date of Filing 01-Dec-2000
Name of Patentee SMITHKLINE BEECHAM BIOLOGICALS S.A
Applicant Address RUE DE 1'INSTITUT 89 B-1330 RIXENSART
Inventors:
# Inventor's Name Inventor's Address
1 VINALS Y DE BASSOLS, CARLOTA SMITHKLINE BEECHAM BIOLOGICALS SA RUE DE 1'INSTITUT 89 B-1330 RIXENSART
PCT International Classification Number C12N 15/31
PCT International Application Number PCT/EP99/03822
PCT International Filing date 1999-05-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 9811945.6 1998-06-03 U.K.
2 9905304.3 1999-03-08 U.K.