Haemophilus influenzae type IV pili
United States Patent: 7,501,131
Issued: March 10, 2009
Inventors: Bakaletz; Lauren
O. (Hilliard, OH), Munson, Jr.; Robert S. (Hilliard, OH)
Children's Hospital, Inc. (Columbus, OH)
Appl. No.: 11/019,005
Filed: December 21, 2004
Web Seminars -- Pharm/Biotech/etc.
The invention described herein relates to
a Haemophilus influenzae (H. influenzae) regulon encoding type IV pili. In
particular, the invention relates to type IV pili from nontypeable H.
influenzae (NTHi) and from H. influenzae strains a, b, c, e and f. The
invention provides isolated H. influenzae pilus polynucleotides and
polypeptides encoded by the polynucleotides as well as polynucleotides and
polypeptides encoded by the polynucleotides involved in the
assembly/disassembly of the structure. The invention also relates to uses
of these polynucleotides and/or polypeptides including methods for
eliciting an immune response to H. influenzae and methods of treating and
preventing H. influenzae related pathological conditions.
Description of the
SUMMARY OF THE INVENTION
The present invention relates to Type IV pilus gene clusters of H.
influenzae, in particular non-typeable H. influenzae (NTHi) and H.
influenzae strains a, b, c, e and f.
Polynucleotides and Polypeptides of the Invention
The present invention provides H. influenzae polynucleotides and
particularly open reading frames from a regulon arranged in two gene
clusters plus one other gene. The regulon includes a gene (pilA) that
encodes the major subunit of a heretofore uncharacterized H. influenzae type
IV pilus. The regulon includes polynucleotides from a gene cluster encoding
pilin polypeptides PilA (major pilin subunit), PilD (leader peptidase), PilB
and PilC (involved in the assembly/disassembly of the pilin structure);
another gene cluster encoding ComA, ComB, ComC, ComD, ComE, and ComF
(involved in competence for transformation and pilus expression); and a gene
encoding PilF (required for type IV pilus biogenesis) (Watson et al, Gene,
49: 56, 1996). In one embodiment, the pilus regulon is that of NTHi H.
influenzae strain 86-028NP.
Polynucleotides encoding the NTHi 86-028NP pilin polypeptides set out in the
following SEQ ID NOs are provided by the invention: PilA polypeptide in SEQ
ID NO: 2, PilB polypeptide in SEQ ID NO: 4, PilC polypeptide in SEQ ID NO:
6, PilD polypeptide in SEQ ID NO: 8, ComA polypeptide in SEQ ID NO: 10, ComB
polypeptide in SEQ ID NO: 12, ComC polypeptide in SEQ ID NO: 14, ComD
polypeptide in SEQ ID NO: 16, ComE polypeptide in SEQ ID NO: 18, ComF
polypeptide in SEQ ID NO: 20 and PilF polypeptide in SEQ ID NO: 22.
Alternative codon usage is thus specifically contemplated by the invention.
In one embodiment, the polynucleotides comprise the NTHi 86-028NP gene
sequences set out in the following SEQ ID NOs which respectively encode the
foregoing polypeptides: pilA in SEQ ID NO: 1, pilB in SEQ ID NO: 3, pilC in
SEQ ID NO: 5, pilD in SEQ ID NO: 7, comA in SEQ ID NO: 9, comB in SEQ ID NO:
11, comC in SEQ ID NO: 13, comD in SEQ ID NO: 15, comE in SEQ ID NO: 17,
comF in SEQ ID NO: 19; and pilF in SEQ ID NO: 21. Each of the polynucleotide
sequences includes a final three nucleotides representing a stop codon.
Also provided are polynucleotides encoding PilA polypeptides from NTHi
clinical isolates 1728MEE, 1729MEE, 3224A, 10548MEE, 1060MEE, 1885MEE,
1714MEE, 1236MEE, 1128MEE and 214NP. The amino acid sequences of these PilA
polypeptides are set out in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42
and 44, respectively. Again, the possibility of alternative codon usage is
specifically contemplated in polynucleotides encoding the polypeptides. In
one embodiment, the polypeptides are respectively encoded by the nucleotide
sequences set out in SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43.
The invention provides for polynucleotides that hybridize under stringent
conditions to (a) the complement of the nucleotide sequences set out in SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37,
39, 41 or 43; (b) a polynucleotide which is an allelic variant of any
polynucleotides recited above; (c) a polynucleotide which encodes a species
homolog of any of the proteins recited above; or (d) a polynucleotide that
encodes a polypeptide comprising a specific domain or truncation of the
polypeptides of the present invention. Type IV pilin polynucleotides from
other non-typeable H. influenzae strains and from H. influenzae strains a,
b, c, e and f are specifically contemplated. These polynucleotides can be
identified and isolated by techniques standard in the art such as
hybridization and polymerase chain reaction using part or all of the
polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25,
27, 29, 31, 33, 35, 37, 39, 41 or 43 as probes or primers, respectively.
The polynucleotides of the invention also include nucleotide sequences that
are substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even
more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the
NTHi polynucleotides recited above.
Included within the scope of the nucleic acid sequences of the invention are
nucleic acid sequence fragments that hybridize under stringent conditions to
the NTHi nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, or complements thereof,
which fragment is greater than about 5 nucleotides, preferably 7
nucleotides, more preferably greater than 9 nucleotides and most preferably
greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides
or more that are selective for (i.e., specifically hybridize to any one of
the polynucleotides of the invention) are contemplated. These nucleic acid
sequence fragments capable of specifically hybridizing to a NTHi
polynucleotide of the invention can be used as probes to detect NTHi
polynucleotides of the invention and/or can differentiate NTHi
polynucleotides of the invention from other bacterial genes, and are
preferably based on unique nucleotide sequences.
The term "stringent" is used herein to refer to conditions that are commonly
understood in the art as stringent. Hybridization stringency is principally
determined by temperature, ionic strength, and the concentration of
denaturing agents such as formamide. Examples of stringent conditions for
hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium
citrate at 65-68.degree. C. or 0.015 M sodium chloride, 0.0015M sodium
citrate, and 50% formamide at 42.degree. C. See Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Laboratory,
(Cold Spring Harbor, N.Y. 1989).
More stringent conditions (such as higher temperature, lower ionic strength,
higher formamide, or other denaturing agent) may also be used, however, the
rate of hybridization will be affected. In instances wherein hybridization
of deoxyoligonucleotides is concerned, additional exemplary stringent
hybridization conditions include washing in 6.times.SSC 0.05% sodium
pyrophosphate at 37.degree. C. (for 14-base oligos), 48.degree. C. (for
17-base oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
Other agents may be included in the hybridization and washing buffers for
the purpose of reducing non-specific and/or background hybridization.
Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1%
sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO.sub.4, (SDS),
ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other
non-complementary DNA), and dextran sulfate, although other suitable agents
can also be used. The concentration and types of these additives can be
changed without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH 6.8-7.4,
however, at typical ionic strength conditions, the rate of hybridization is
nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A
Practical Approach, Ch. 4, IRL Press Limited (Oxford, England).
Hybridization conditions can be adjusted by one skilled in the art in order
to accommodate these variables and allow DNAs of different sequence
relatedness to form hybrids.
As noted above, polynucleotides contemplated by the present invention are
not limited to the specific polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, but also
include, for example, allelic and species variations thereof. Allelic and
species variations can be routinely determined by comparing the sequence
provided in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29,
31, 33, 35, 37, 39, 41 or 43, preferably the open reading frames therein, a
representative fragment thereof, or a nucleotide sequence at least 90%
identical, preferably 95% identical, to the open reading frames within SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37,
39, 41 or 43 with a sequence from another isolate of the same species or
another species. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited to, the
GCG program package, including GAP (Devereux et al., Nuc. Acid. Res., 12:
387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.),
BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410,
1990). The BLASTX program is publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul
et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well
known Smith-Waterman algorithm may also be used to determine identity.
Polynucleotides of the invention may be isolated from natural sources or may
be synthesized by standard chemical techniques, e.g., the phosphotriester
method described in Matteucci et al., J. Am Chem Soc., 103: 3185 (1981).
Antisense polynucleotides complementary to the polynucleotides encoding the
pilus polypeptides of the invention are also provided.
Polypeptides of the invention include pilin polypeptides PilA, PilD, PilB,
PilC, ComA, ComB, ComC, ComD, ComE, ComF and PilF. In one embodiment the
polypeptides comprise the NTHi 86-028NP amino acid sequences respectively
set out in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. Polypeptides
of the invention also include PilA polypeptides set out in SEQ ID NOs: 26,
28, 30, 32, 34, 36, 38, 40, 42 or 44. In additional embodiments, the Type IV
pilin polypeptides of the invention are those of other non-typeable H.
influenzae strains and from H. influenzae strains a, b, c, e and f.
Polypeptides of the invention specifically include peptide fragments (i.e.,
peptides) that retain one or more biological or immunogenic properties of a
full length polypeptide of the invention. In one embodiment PilA peptide
fragments provided by the invention are designated TfpQ2, TFPQ3, TfpQ4 and
OLP3 and respectively comprise amino acids 35 through 68 of SEQ ID NO: 2,
amino acids 69 through 102 of SEQ ID NO: 2, amino acids 103 through 137 of
SEQ ID NO: 2, and amino acids 21 through 35 of SEQ ID NO: 2.
The invention also provides for polypeptides with one or more conservative
amino acid substitutions that do not affect the biological and/or
immunogenic activity of the polypeptide. Alternatively, the polypeptides of
the invention are contemplated to have conservative amino acids
substitutions which may or may not alter biological activity. The term
"conservative amino acid substitution" refers to a substitution of a native
amino acid residue with a nonnative residue, including naturally occurring
and nonnaturally occurring amino acids, such that there is little or no
effect on the polarity or charge of the amino acid residue at that position.
For example, a conservative substitution results from the replacement of a
non-polar residue in a polypeptide with any other non-polar residue.
Further, any native residue in the polypeptide may also be substituted with
alanine, according to the methods of "alanine scanning mutagenesis".
Naturally occurring amino acids are characterized based on their side chains
as follows: basic: arginine, lysine, histidine; acidic: glutamic acid,
aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine,
tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine,
alanine, valine, proline, methionine, leucine, norleucine, isoleucine
General rules for amino acid substitutions are set forth in Table 1 below
-- see Original Patent.
The invention also provides
variants of the polypeptides of the present invention (e.g., a polypeptide
exhibiting at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%,
91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically
at least about 98%, or most typically at least about 99% amino acid identity
to a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22)
that retain biological and/or immunogenic activity.
The invention contemplates that polynucleotides of the invention may be
inserted in a vector for amplification or expression. For expression, the
polynucleotides are operatively linked to appropriate expression control
sequences such as promoter and polyadenylation signal sequences. Further
provided are host cells comprising polynucleotides of the invention.
Exemplary prokaryotic host cells include bacteria such as E. coli, Bacillus,
Streptomyces, Pseudomonas, Salmonella and Serratia. Methods of producing
polypeptides of the invention by growing the host cells and isolating
polypeptide from the host cells or growth medium are specifically
contemplated. One or more polynucleotides from the pilus regulon may be
expressed in a host cell. For example, expression of the pilA gene alone and
expression of multiple polynucleotides from the pilus regulon in order to
affect assembly of the native pili structure are both specifically
contemplated. Alternatively, polypeptides of the invention can be prepared
by chemical synthesis using standard means. Particularly convenient are
solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v. 2,
Academic Press, New York, p. 255). Automated solid phase synthesizers are
commercially available. In addition, modifications in the sequence are
easily made by substitution, addition or omission of appropriate residues.
For example, a cysteine residue may be added at the carboxy terminus to
provide a sulfhydryl group for convenient linkage to a carrier protein, or
spacer elements, such as an additional glycine residue, may be incorporated
into the sequence between the linking amino acid at the C-terminus and the
remainder of the peptide.
The term "isolated" refers to a substance removed from, and essentially free
of, the other components of the environment in which it naturally exists.
For example, a polypeptide is separated from other cellular proteins or a
DNA is separated from other DNA flanking it in a genome in which it
The invention, provides antibodies which bind to antigenic epitopes unique
to (i.e., are specific for) H. influenzae pilus polypeptides of the
invention. Also provided are antibodies which bind to ant igenic epiropes
common among multiple H. influenzae subtypes but unique with respect to any
other antigenic epitopes. The antibodies may be polyclonal antibodies,
monoclonal antibodies, antibody fragments which retain their ability to bind
their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chain
antibodies and human or humanized antibodies. Antibodies may be generated by
techniques standard in the art using pilin polypeptide(s) of the invention
or host cells expressing pilin polypeptide(s) of the invention as antigens.
The present invention provides for antibodies specific for the pilin
polypeptides of the present invention and fragments thereof, which exhibit
the ability to kill both H. influenzae bacteria and to protect humans from
infection. The present invention also provides for antibodies specific for
the polypeptides of the invention which reduce the virulence, inhibit
adherence, inhibit biofilm formation, inhibit twitching motility, inhibit
cell division, and/or inhibit penetration into the epithelium of H.
influenzae bacteria and/or enhance phagocytosis of the H. influenzae
In vitro complement mediated bactericidal assay systems (Musher et al.,
Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51:
31-38, 1972) may be used to measure the bactericidal activity of anti-pilus
It is also possible to confer short-term protection to a host by passive
immunotherapy via the administration of pre-formed antibody against an H.
influenzae polypeptide of the invention. Thus, antibodies of the invention
may be used in passive immunotherapy. Human immunoglobulin is preferred in
human medicine because a heterologous immunoglobulin may provoke an immune
response to its foreign immunogenic components. Such passive immunization
could be used on an emergency basis for immediate protection of unimmunized
individuals subject to special risks.
In another embodiment, antibodies of the invention may be used in the
production of anti-idiotypic antibody, which in turn can be used as an
antigen to stimulate an immune response against pilin epitopes.
Methods for Eliciting an Immune Response and Compositions Therefor
The invention contemplates methods of eliciting in an individual an immune
response to one or more H. influenzae type IV pilus polypeptides. In certain
embodiments, the methods elicit an immune response to the PilA protein.
These methods elicit one or more immune responses, including but not limited
to, immune responses which inhibit bacterial replication, immune responses
which block H. influenzae adherence to cells, immune responses which prevent
H. influenzae twitching and immune responses which prevent biofilm
formation. In one embodiment, the methods comprise a step of administering
an immunogenic dose of a composition comprising one or more polypeptides of
the invention. In another embodiment, the methods comprise administering an
immunogenic dose of a composition comprising a cell expressing one or more
polypeptides of the invention. In yet another embodiment, the methods
comprise administering an immunogenic dose of a composition comprising one
or more polynucleotides encoding one or more polypeptides of the invention.
The polynucleotide may be a naked polynucleotide not associated with any
other nucleic acid or may be in a vector such as a plasmid or viral vector
(e.g., adeno-associated virus vector or adenovirus vector). The methods may
be used in combination in a single individual. The methods may be used prior
or subsequent to H. influenzae infection of an individual.
In one embodiment of methods of the invention, a composition of the
invention is administered as a priming dose followed by one or more booster
doses. Co-administration of proteins or polypeptides that beneficially
enhance the immune response such as cytokines (e.g., IL-2, IL-12, GM-CSF),
cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules is also
An "immunogenic dose" of a composition of the invention is one that
generates, after administration, a detectable humoral (antibody) and/or
cellular (T cell) immune response in comparison to the immune response
detectable before administration or in comparison to a standard immune
response before administration. The invention contemplates that the immune
response resulting from the methods may be protective and/or therapeutic. In
a preferred embodiment, the antibody and/or T cell immune response protects
the individual from H. influenzae infection, particularly infection of the
middle ear and/or the nasopharynx or lower airway. In this use, the precise
dose depends on the patient's state of health and weight, the mode of
administration, the nature of the formulation, etc., but generally ranges
from about 1.0 .mu.g to about 5000 .mu.g per 70 kilogram patient, more
commonly from about 10 to about 500 .mu.g per 70 kg of body weight.
Humoral immune response may be measured by many well known methods, such as
Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and
Hemagglutination Inhibition Assay (HAI). In particular, SRID utilizes a
layer of a gel, such as agarose, containing the immunogen being tested. A
well is cut in the gel and the serum being tested is placed in the well.
Diffusion of the antibody out into the gel leads to the formation of a
precipitation ring whose area is proportional to the concentration of the
antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked
Immunoassay), is used to determine total antibodies in the sample. The
immunogen is adsorbed to the surface of a microtiter plate. The test serum
is exposed to the plate followed by an enzyme linked immunoglobulin, such as
IgG. The enzyme activity adherent to the plate is quantified by any
convenient means such as spectrophotometry and is proportional to the
concentration of antibody directed against the immunogen present in the test
sample. HAI utilizes the capability of an immunogen such as viral proteins
to agglutinate chicken red blood cells (or the like). The assay detects
neutralizing antibodies, i.e., those antibodies able to inhibit
hemagglutination. Dilutions of the test serum are incubated with a standard
concentration of immunogen, followed by the addition of the red blood cells.
The presence of neutralizing antibodies will inhibit the agglutination of
the red blood cells by the immunogen. Tests to measure cellular immune
response include determination of delayed-type hypersensitivity or measuring
the proliferative response of lymphocytes to target immunogen.
The invention correspondingly provides compositions suitable for eliciting
an immune response to pilus polypeptides of the invention. As noted above,
the compositions comprise one or more pilus polypeptides, cells expressing
one or more polypeptides, or one or more polynucleotides encoding one or
more pilus polypeptides. The compositions may also comprise other
ingredients such as carriers and adjuvants.
In compositions of the invention, a pilus polypeptide may be fused to
another protein when produced by recombinant methods. In one embodiment, the
other protein may not, by itself, elicit antibodies, but it stabilizes the
first protein and forms a fusion protein retaining immunogenic activity. In
another embodiment, the fusion protein comprises another protein that is
immunogenic, such as Glutathione-S-transferase (GST) or beta-galactosidase,
relatively large co-proteins which solubilize the fusion protein and
facilitate production and purification thereof. The other protein may act as
an adjuvant in the sense of providing a generalized stimulation of the
immune system. The other protein may be fused to either the amino or carboxy
terminus of the NTHi protein of the invention.
In other compositions of the invention, pilus polypeptides may be otherwise
linked to carrier substances. Any method of creating such linkages known in
the art may be used. Linkages can be formed with heterobifunctional agents
that generate a disulfide link at one functional group end and a peptide
link at the other, such as a disulfide amide forming agent, e.g.,
N-succidimidyl-3-(2-pyridyldithio)proprionate (SPDP) (See, e.g., Jansen et
al., Immun. Rev. 62:185, 1982) and bifunctional coupling agents that form a
thioether rather than a disulfide linkage such as reactive esters of
6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid,
4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, and
coupling agent which activate carboxyl groups by combining them with
succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as
succinimmidyl 4-(N-maleimido-methyl)cyclohexane-1-carobxylate (SMCC).
The pilus polypeptides may be formulated as neutral or salt forms.
Pharmaceutically acceptable salts, include the acid addition salts (formed
with the free amino groups of the peptide) and which are formed with
inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the
free carboxyl groups may also be derived from inorganic bases such as, e.g.,
sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
Compositions of the invention may further comprise adjuvants. Known
adjuvants include, for example, emulsions such as Freund's Adjuvants and
other oil emulsions, Bordetella pertussis, MF59, purified saponin from
Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and
alum, calcium phosphate, (and other metal salts), gels such as aluminum
hydroxide salts, mycobacterial products including muramyl dipeptides, solid
materials, particles such as liposomes and virosomes. Examples of natural
and bacterial products known to be used as adjuvants include monophosphoryl
lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174
which is a lipid A derivative from E. coli, holotoxins such as cholera toxin
(CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin
(LT) of E. coli or one of its derivatives, and CpG oligonucleotides.
Adjuvant activity can be affected by a number of factors, such as carrier
effect, depot formation, altered lymphocyte recirculation, stimulation of
T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of
Compositions of the invention are typically formulated as injectables,
either as liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid prior to injection may also be prepared. The
preparation may also be emulsified. The active immunogenic ingredient is
often mixed with excipients, which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, e.g., water,
saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering agents, or
adjuvants, which enhance the effectiveness of the vaccine. The vaccines are
conventionally administered parenterally, by injection, for example, either
subcutaneously or intramuscularly.
Additional formulations which are suitable for other modes of administration
include suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include, for example,
polyalkalene glycols or triglycerides; such suppositories may be formed from
mixtures containing the active ingredient in the range of 0.5% to 10%,
preferably 1-2%. Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and
the like. These compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or powders and
contain 10%-95% of active ingredient, preferably 25-70%.
Compositions may also be administered through transdermal routes utilizing
jet injectors, microneedles, electroporation, sonoporation,
microencapsulation, polymers or liposomes, transmucosal routes and
intranasal routes using nebulizers, aerosols and nasal sprays.
Microencapsulation using natural or synthetic polymers such as starch,
alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic
microspheres, polycaprolactones, polyorthoesters, polyanhydrides and
polyphosphazenes polyphosphatazanes are useful for both transdermal and
transmucosal administration. Polymeric complexes comprising synthetic poly-omithate,
poly-lysine and poly-arginine or amphipathic peptides are useful for
transdermal delivery systems. In addition, due to their amphipathic nature,
liposomes are contemplated for transdermal, transmucosal and intranasal
vaccine delivery systems. Common lipids used for vaccine delivery include
N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl sulfate (DOTAP),
dioleyloxy-propyl-trimethylammonium chloride DOTMA,
dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE),
dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N',N-dimethylaminoethane)
carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and
liposomes will enhance up-take of the liposomes through the skin. These
helper lipids include, dioleoyl phosphatidylethanolamine (DOPE),
dilauroylphosphatidylethanolamine (DLPE), dimyristoyl
phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE).
In addition, triterpenoid glycosides or saponins derived from the Chilean
soap tree bark (Quillaja saponaria) and chitosan (deacetylated chitan) have
been contemplated as useful adjuvants for intranasal and transmucosal
Formulations may be presented in unit-dose or multi-dose containers, for
example, sealed ampules and vials and may be stored in a freeze-dried
condition requiring only the addition of the sterile liquid carrier
immediately prior to use.
Methods of Inhibiting H. influenzae
Alternatively, the invention includes methods of inhibiting H. influenzae
type IV pili function in an individual. The methods comprise administering
to the individual, for example, one or more antibodies of the invention; one
or more polypeptides of the invention; one or more antisense polynucleotides
of the invention; one or more RNAi molecules; and/or one or more small
molecules, in an amount that inhibits function of the pili. In vitro assays
may be used to demonstrate the ability to inhibit pili function. Embodiments
of these methods include, for example, methods using inhibitors of pilus
polyepeptide synthesis and/or pilus assembly, inhibitors of adherence
mediated via type IV pili, inhibitors that disrupt existing biofilms
mediated by type IV pili, and inhibitors of twitching.
Inhibition is contemplated for any pathological condition involving H.
influenzae, for example, OM, pneumonia, sinusitis, septicemia, endocarditis,
epiglottitis, septic arthritis, meningitis, postpartum and neonatal
infections, postpartum and neonatal sepsis, acute and chromic salpingitis,
epiglottis, pericardis, cellulitis, osteomyelitis, endocarditis,
cholecystitis, intraabdominal infections, urinary tract infection,
mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric
fever, occult bacteremia and exacerbation of underlying lung diseases such
as chronic bronchitis, bronchietasis and cystic fibrosis.
Compositions comprising inhibitors of H. influenzae type IV pili function
are provided. The compositions may consist of one of the foregoing active
ingredients alone, may comprise combinations of the foregoing active
ingredients or may comprise additional active ingredients used to treat
bacterial infections. As discussed above, the compositions may comprise one
or more additional ingredients such as pharmaceutically effective carriers.
Also as discussed above, dosage and frequency of the administration of the
compositions are determined by standard techniques and depend, for example,
on the weight and age of the individual, the route of administration, and
the severity of symptoms. Administration of the pharmaceutical compositions
may be by routes standard in the art, for example, parenteral, intravenous,
oral, buccal, nasal, pulmonary, rectal, intranasal, or vaginal.
Methods of the invention may be demonstrated in a chinchilla model widely
accepted as an experimental model for OM. In particular, a chinchilla model
of NTHi-induced OM has been well characterized (Bakaletz et al., J. Infect.
Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol.,
4: 223-225, 1997; Suzuki and Bakaletz, Infect. Immun., 62: 1710-1718, 1994;
Mason et al., Infect. Immun., 71:3454-3462, 2003), and has been used to
determine the protective efficacy of several NTHi outer membrane proteins,
combinations of outer membrane proteins, chimeric synthetic peptide vaccine
components, and adjuvant formulations against OM (Bakaletz et al., Vaccine,
15: 955-961, 1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999;
Kennedy et al., Infect. Immun., 68: 2756-2765, 2000; Kyd et al., Infect.
Immun., 66:2272-2278, 2003; Novotny and Bakaletz, J. Immunol., 171,
In the model, adenovirus predisposes chinchillas to H. influenzae-induced OM
media, which allowed for the establishment of relevant cell, tissue and
organ culture systems for the biological assessment of NTHi (Bakaletz et
al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et al., Infect. Immunity 62:
1710-8, 1994). Adenovirus infection alone has been used to assess the
transudation of induced serum antibodies into the tympanum (Bakaletz et al.,
Clin. Diagnostic Lab Immunol., 4(2): 223-5, 1997) and has been used as a
co-pathogen with NTHi, to determine the protective efficacy of several
active and passive immunization regimens targeting various NTHi outer
membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine
components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz
et al., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect. Immun.,
68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4): 2119-28, 2000;
Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).
Methods of Detecting H. influenzae Bacteria
Also provided by the invention are methods for detecting bacteria in an
individual. In one embodiment, the methods comprise detecting pili
polynucleotides of the invention in a biological sample using primers or
probes that specifically bind to the polynucleotides. Detection of the
polynucleotide may be accomplished by numerous techniques routine in the art
involving, for example, hybridization and/or PCR. In another embodiment, the
methods comprise detecting pili polypeptides of the invention in a
biological sample using antibodies of the invention that specifically bind
to the polypeptides. The antibodies may be used in any immunoassay system
known in the art including, but not limited to, radioimmunoassays, ELISA
assays, sandwich assays, precipitin reactions, gel diffusion precipitin
reactions, immunodiffusion assays, agglutination assays, fluorescent
immunoassays, protein A immunoassays and immunoelectrophoresis assays.
Biological samples to be utilized in the methods include, but are not
limited to, blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic
fluid and cerebrospinal fluid.
Claim 1 of 16 Claims
1. An isolated polypeptide comprising an
amino acid sequence encoded by a nucleotide sequence selected from the
group consisting of pilA SEQ ID NO: 1, pilB SEQ ID NO: 3, pilC SEQ ID NO:
5, pilD SEQ ID NO: 7, comA SEQ ID NO: 9, comB SEQ ID NO: 11, comC SEQ ID
NO: 13, comD SEQ ID NO: 15, comE SEQ ID NO: 17, comF SEQ ID NO: 19, pilF
SEQ ID NO: 21, pilA SEQ ID NO: 33, pilA SEQ ID NO: 35, pilA SEQ ID NO: 37,
pilA SEQ ID NO: 39, pilA SEQ ID NO: 41and pilA SEQ ID NO: 43.
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