U.S. patent application number 13/488251 was filed with the patent office on 2013-06-20 for compositions and methods related to p6.
This patent application is currently assigned to ROCHESTER GENERAL HOSPITAL RESEARCH INSTITUTE. The applicant listed for this patent is JANET CASEY, RAVINDER KAUR, M. NADEEM KHAN, LEA MICHEL, MICHAEL PICHICHERO, SHARAD SHARMA. Invention is credited to JANET CASEY, RAVINDER KAUR, M. NADEEM KHAN, LEA MICHEL, MICHAEL PICHICHERO, SHARAD SHARMA.
Application Number | 20130156803 13/488251 |
Document ID | / |
Family ID | 46397619 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130156803 |
Kind Code |
A1 |
PICHICHERO; MICHAEL ; et
al. |
June 20, 2013 |
COMPOSITIONS AND METHODS RELATED TO P6
Abstract
Disclosed are compositions and methods related to vaccination
for AOM and children prone to AOM.
Inventors: |
PICHICHERO; MICHAEL;
(ROCHESTER, NY) ; KHAN; M. NADEEM; (ROCHESTER,
NY) ; KAUR; RAVINDER; (PITTSFORD, NY) ;
SHARMA; SHARAD; (ROCHESTER, NY) ; CASEY; JANET;
(PITTSFORD, NY) ; MICHEL; LEA; (ROCHESTER,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PICHICHERO; MICHAEL
KHAN; M. NADEEM
KAUR; RAVINDER
SHARMA; SHARAD
CASEY; JANET
MICHEL; LEA |
ROCHESTER
ROCHESTER
PITTSFORD
ROCHESTER
PITTSFORD
ROCHESTER |
NY
NY
NY
NY
NY
NY |
US
US
US
US
US
US |
|
|
Assignee: |
ROCHESTER GENERAL HOSPITAL RESEARCH
INSTITUTE
|
Family ID: |
46397619 |
Appl. No.: |
13/488251 |
Filed: |
June 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61493437 |
Jun 4, 2011 |
|
|
|
Current U.S.
Class: |
424/190.1 ;
424/256.1; 435/188; 435/7.92; 530/300 |
Current CPC
Class: |
A61K 39/102 20130101;
A61K 2039/57 20130101; G01N 33/6854 20130101 |
Class at
Publication: |
424/190.1 ;
530/300; 424/256.1; 435/188; 435/7.92 |
International
Class: |
A61K 39/102 20060101
A61K039/102; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government Support under RO1
08671 by the National Institutes of Health and National Institute
on Deafness and Other Communication Disorders. The Government has
certain rights in the invention.
Claims
1. An epitope for an immunization against Nontypeable Haemophilus
influenzae (NTHi), selected from the group of peptides of 3B9, 7F3
and 4G4, or mixtures thereof.
2. The epitope of claim 1, reactive with anti-P6 polyclonal
antibodies.
3. The epitopes of claim 2, in combination with a pharmaceutical
carrier for administration to a subject.
4. The epitopes of claim 3, in an effective concentration for
administration to a subject to neutralize Nontypeable Haemophilus
influenzae (NTHi).
5. The epitopes of claim 1, further comprising a pharmaceutical
carrier for administration to a patient, wherein the carrier and
concentration of sequences elicit an immune response when
administered to a subject.
6. The epitopes of claim 1 labeled with a compound selected from
the group consisting of dyes, fluorescent labels, chemiluminescent
labels, enzymes, and radioactive labels.
7. The epitopes of claim 1 immobilized onto a substrate.
8. A method for screening patients for Nontypeable Haemophilus
influenzae (NTHi) infection comprising reacting a biological sample
with the epitopes of claim 1.
9. The method of claim 8 wherein the epitopes are labeled with a
compound selected from the group consisting of dyes, fluorescent
labels, chemiluminescent labels, enzymes, and radioactive
labels.
10. The method of claim 8 wherein the epitopes are immobilized onto
a substrate.
11. The method of claim 10, further comprising predicting the
prognosis of the patient based on the reactivity of the patient
sample with the epitopes.
12. A method for treating patients for Nontypeable Haemophilus
influenzae (NTHi) comprising administering to the patient an
epitope of claim 1, or mixtures thereof, in an amount effective to
inhibit a Nontypeable Haemophilus influenzae (NTHi) infection.
13. The method of claim 12 wherein the epitope acts as a vaccine
against Nontypeable Haemophilus influenzae (NTHi) infection.
14. The method of claim 13, wherein the subject has an Nontypeable
Haemophilus influenzae (NTHi) ear infection.
15. The method of claim 12, wherein the subject is prone to ear
infections.
16. A vaccine for inhibiting infection comprising the epitope of
claim 1, in combination with an antigenic pharmaceutical
carrier.
17. A method for vaccinating a host against a Nontypeable
Haemophilus influenzae (NTHi) infection comprising providing the
epitope of claim 1 in combination with an antigenic pharmaceutical
carrier.
18. The epitope of claim 1, wherein the epitope is at a
concentration of at least 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5,
3.0, 5, 7, or 10 fold the P6 and P6 epitope of a vaccine having P6,
protein D, and OMP24 present in them.
19. A vaccine comprising the epitope of claim 18.
20. The vaccine of claim 19, further comprising P6 at a
concentration of at least 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5,
3.0, 5, 7, or 10 fold the P6 and P6 epitope of a vaccine having P6,
protein D, and OMP24 present in them.
21. A vaccine comprising P6 at a concentration of at least 1.1,
1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5, 3.0, 5, 7, or 10 fold the P6 and
P6 epitope of a vaccine having P6, protein D, and OMP24 present in
them.
22. A vaccine further comprising the epitope of claim 18.
23. A vaccine comprising P6, or an epitope of claim 1, or mixture
there of, wherein the vaccine comprises a concentration of antigen
greater than 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5, 3.0, 5, 7, or
10 fold the P6 and P6 epitope antigen of a vaccine having P6,
protein D, and OMP24 present in them.
24. The vaccine as claimed in claim 8, wherein the vaccine is in a
capsular form.
25. A method of vaccination, said method comprising step of
administering therapeutically effective dose of the vaccine of
claim 23 to a subject in need thereof.
26. The method of claim 25, wherein the subject has Nontypeable
Haemophilus influenzae (NTHi).
27. The method of claim 25, wherein the subject is prone to have
Nontypeable Haemophilus influenzae (NTHi).
28. The method of claim 25, wherein the vaccine is administered
through an oral route or intra peritoneal route.
29. The method of claim 25, wherein the vaccine is administered in
capsular form.
30. The method of claim 25, wherein the vaccine is administered at
a dosage ranging from 0.01 mg/ml/kg to 100 mg/ml/kg.
31. The method of claim 25, wherein the vaccine is capable of
inhibiting an Nontypeable Haemophilus influenzae (NTHi) infection
in a subject prone to Nontypeable Haemophilus influenzae (NTHi), to
a greater extent than a vaccine comprising Protein D or OMP26.
32. A kit for vaccination, said kit comprising a vaccine of claim
22 in a deliverable form.
33. A vaccine comprising P6 and Protein D, epitopes of P6 or
Protein D, or mixtures thereof, but not OMP26 of non typeable
Haemophilus influenzae (NTHi).
34. The vaccine of claim 33, wherein the vaccine consists of P6 or
P6 epitopes.
35. A method of treating a child, comprising identifying a child
wherein the child is prone to having acute otitis media (AOM) and
administering the vaccine of claim 34 to the child.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 61/493,437, filed Jun. 4, 2011. Application No.
61/493,437, filed Jun. 4, 2011, is hereby incorporated herein by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The Sequence Listing submitted Jun. 4, 2012 as a text file
named "RGH.sub.--101_AMD_AFD_Sequence_Listing.txt," created on Jun.
4, 2012, and having a size of 1,725 bytes, is hereby incorporated
by reference pursuant to 37 C.F.R. .sctn.1.52(e)(5)
FIELD OF THE INVENTION
[0004] The field of the invention is compositions, methods and kits
related to therapies Acute Otitis Media (AOM) and other respiratory
diseases and disorders caused by the bacteria nontypeable
Haemophilus influenzae (NTHi).
BACKGROUND OF THE INVENTION
[0005] Acute Otitis Media (AOM) is the most common infectious
disease among children to cause parents to seek medical care for
their child. Children receive antibiotics to treat AOM which
increases the emergence of antibiotic resistant bacteria. Temporary
hearing loss is the most common complication; rarely there are
intracranial complications. WHO estimates that 51,000 deaths/year
are attributable to AOM in children younger than 5 years old and
that chronic AOM (occurring in 65-330 million people) is the major
cause of hearing loss in developing countries. Nontypeable
Haemophilus influenzae (NTHi) bacteria accounts for 40-60% of AOM
and recurrent AOM. A similar percentage of cases of acute sinusitis
and rhinosinusitis, acute exacerbations of chronic bronchitis and
acute pneumonia (in the developing world) are caused by NTHi.
[0006] Otitis prone (OP) children are defined as children with
recurrent AOM, with at least 3 episodes in 6 months or 4 episodes
in a 12-month time span. Each episode of AOM is typically followed
by 4-12 weeks of otitis media with effusion (OME) during which time
the child has diminished hearing and this often leads to temporary
delayed speech and language development and can be associated with
permanent hearing loss. Non otitis prone (NOP) children experience
no ear infections or few ear infections, not meeting the OP
definition. In the US alone, the economic burden of otitis media
exceeded $5 billion/year in 1997 in medical treatment, surgical
management, and loss of income for working parents. Thus, the
impact on health costs and on lifestyle for the child and parents
is very meaningful. OP children eventually lose their propensity to
experience AOM, usually by age 5 years.
[0007] Due to the large costs related to AOM, there is a need for
an effective preventive or prophylactic treatment. OP children do
not always generate an efficient immune response and thus a vaccine
that generates an immune response that efficiently destroys the
pathogen is needed.
[0008] The disclosed invention provides a vaccine that produces
humoral and cell mediated responses to NTHi for preventing ear
infections, sinus infections, acute exacerbations of bronchitis and
pneumonia. The invention provides epitopes that are of importance
for generating an effective immune response to NTHi.
[0009] The invention further provides methods of vaccinating
individuals with an effective NHTi vaccine.
SUMMARY OF THE INVENTION
[0010] Disclosed are compositions and methods related to Acute
Otitis Media (AOM). For example, disclosed are epitopes for an
immunization against Nontypeable Haemophilus influenzae (NTHi),
selected from the group of peptides of 3B9, 7F3 and 4G4, or
mixtures thereof.
[0011] Also disclosed are vaccines comprising one or more of the
epitopes. Also disclosed are vaccines for inhibiting infection
comprising the epitope of claim 1, in combination with an antigenic
pharmaceutical carrier. Also disclosed are vaccines comprising P6
at a concentration of at least 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0,
2.5, 3.0, 5, 7, or 10 fold the P6 and P6 epitope of a vaccine
having P6, protein D, and OMP24 present in them. Also disclosed are
vaccines comprising P6, or an epitope of claim 1, or mixture there
of, wherein the vaccine comprises a concentration of antigen
greater than 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5, 3.0, 5, 7, or
10 fold the P6 and P6 epitope antigen of a vaccine having P6,
protein D, and OMP24 present in them. Also disclosed are vaccines
comprising P6 and Protein D, epitopes of P6 or Protein D, or
mixtures thereof, but not OMP26 of non typeable Haemophilus
influenzae (NTHi).
[0012] Also disclosed are methods for screening patients for
Nontypeable Haemophilus influenzae (NTHi) infection comprising
reacting a biological sample with the epitopes of claim 1. Also
disclosed are methods for treating patients for Nontypeable
Haemophilus influenzae (NTHi) comprising administering to the
patient an epitope of claim 1, or mixtures thereof, in an amount
effective to inhibit a Nontypeable Haemophilus influenzae (NTHi)
infection. Also disclosed are methods for vaccinating a host
against a Nontypeable Haemophilus influenzae (NTHi) infection
comprising providing the epitope of claim 1 in combination with an
antigenic pharmaceutical carrier. Also disclosed are methods of
vaccination, said method comprising step of administering
therapeutically effective dose of the vaccine of claim 23 to a
subject in need thereof. Also disclosed are methods of treating a
child, comprising identifying a child wherein the child is prone to
having acute otitis media (AOM) and administering the vaccine of
claim 34 to the child.
[0013] Also disclosed are kits for vaccination, the kit comprising
a vaccine in a deliverable form.
[0014] The epitopes can be reactive with anti-P6 polyclonal
antibodies. The epitopes can be used in combination with a
pharmaceutical carrier for administration to a subject. The
epitopes can be used in an effective concentration for
administration to a subject to neutralize Nontypeable Haemophilus
influenzae (NTHi). The epitopes can further comprise a
pharmaceutical carrier for administration to a patient, wherein the
carrier and concentration of sequences elicit an immune response
when administered to a subject. The epitopes can be labeled with a
compound selected from the group consisting of dyes, fluorescent
labels, chemiluminescent labels, enzymes, and radioactive labels.
The epitopes can be immobilized onto a substrate. The epitopes can
be used at a concentration of at least 1.1, 1.2, 1.3, 1.5, 1.7,
1.9, 2.0, 2.5, 3.0, 5, 7, or 10 fold the P6 and P6 epitope of a
vaccine having P6, protein D, and OMP24 present in them.
[0015] The vaccines can comprise one or more of the epitope. The
vaccines can comprise one or more of the epitopes and P6 at a
concentration of at least 1.1, 1.2, 1.3, 1.5, 1.7, 1.9, 2.0, 2.5,
3.0, 5, 7, or 10 fold the P6 and P6 epitope of a vaccine having P6,
protein D, and OMP24 present in them. The vaccines can be in a
capsular form. The vaccines can consist of P6 or P6 epitopes.
[0016] The methods can use epitopes labeled with a compound
selected from the group consisting of dyes, fluorescent labels,
chemiluminescent labels, enzymes, and radioactive labels. The
methods can use epitopes immobilized onto a substrate. The methods
can further comprise predicting the prognosis of the patient based
on the reactivity of the patient sample with the epitopes. The
methods can use epitopes that act as a vaccine against Nontypeable
Haemophilus influenzae (NTHi) infection. The methods can treat
patients and subjects having Nontypeable Haemophilus influenzae
(NTHi), having a Nontypeable Haemophilus influenzae (NTHi) ear
infection, and/or prone to having Nontypeable Haemophilus
influenzae (NTHi).
[0017] The methods can administer the vaccines through an oral
route or intra peritoneal route. The methods can administer the
vaccines in capsular form. The methods can administer the vaccines
at a dosage ranging from 0.01 mg/ml/kg to 100 mg/ml/kg. The methods
can use vaccines capable of inhibiting a Nontypeable Haemophilus
influenzae (NTHi) infection in a subject prone to Nontypeable
Haemophilus influenzae (NTHi), to a greater extent than a vaccine
comprising Protein D or OMP26.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A, 1B, 1C and 1D are graphs of antibody titers. FIG.
1A is a graph of the bactericidal titers against homologous and
heterologous NTHi strains elicited in children with AOM. FIG. 1B is
a graph of the comparison of anti P6 ELISA titers in bactericidal
and non bactericidal sera elicited in children with AOM. FIG. 1C is
a graph of the comparison of anti Protein D ELISA titers in
bactericidal and non bactericidal sera elicited in children with
AOM. FIG. 1D is a graph of the whole cell ELISA titers against
bactericidal and non bactericidal sera elicited in children with
AOM.
[0019] FIG. 2A is a graph of the NTHi whole cell antibody titers
against homologous and heterologous NTHi strains in bactericidal
sera elicited in children with AOM. FIGS. 2B and 2C are graphs of
the correlation of anti protein D and P6 IgG titers and
bactericidal titers, respectively.
[0020] FIGS. 3A, 3B, 3C and 3D are graphs of percent frequencies of
NTHi antigen specific memory CD4+ T cells. A) IFN-.gamma., B) IL-4,
C) IL-2, and D) IL-17A, in the circulation of OP and NOP children
against NTHi antigens (P6, OMP26, and Protein D). Bar graphs
represent mean percentage values of CD69+ CD4+ T cells gated on
CD45RAlo following antigen stimulation +/-SE. *p<0.05 (n=10 each
group; OP: average age 14.5 months and NOP: average age 8.2
months).
[0021] FIG. 4 is a graph showing inflammasome activation is higher
in OP than NOP children. Expression of NLRP3 was determined in
nasal cell pellets of OP (n=3) and NOP (n=5) children (<2 years)
with AOM. Relative fold change of OP over NOP shown. +/-SEM from
children 915 months old. 18S used as a calibrator.
[0022] FIG. 5 is a graph showing higher bacterial receptor
upregulation in the nasal mucosa of OP children. The Spn expression
pattern of nasal cell pellets was assessed by RT-PCR. Data shown
represents average fold change of expression of OP over NOP
children +/-SD. (n=5 each group with average age of 9 months)
[0023] FIG. 6 is a graph showing that proinflammatory cytokine
expression diverges in NOP and OP children. Expression of
proinflammatory genes were determined in nasal cell pellets of OP
(n=4) and NOP (n=5) children (<2 years) with AOM. Relative fold
change of OP over NOP shown. +/-SEM 6-15 months old in each group.
*p<0.05 for .DELTA.C.sub.T values **p=0.09. 18S used as a
calibrator.
[0024] FIG. 7 is a graph showing divergent TLR expression in OP and
NOP children. Expression of TLR2 and TLR4 were determined in the
nasal wash of OP (n=4) and NOP (n=5) children (<2 years) during
AOM. Relative fold change of OP over NOP shown. +/-SEM. *p=0.01 for
.DELTA.C.sub.T values. 18S used as a calibrator.
[0025] FIG. 8 is a comparison of differentially expressed immune
response related genes obtained from PBMCs of children (18 months
old) with Spn or NTHi induced AOMs expressed relative to their
respective healthy control visits. Genes regulated greater than 2
fold are represented.
[0026] FIG. 9 shows nasopharyngeal and/or oropharyngeal
colonization episodes represented by a closed circle at visits 1-7
at 6, 9, 12, 15, 18, 24, and 30 months of age. AOM episodes are
represented by a closed triangle.
[0027] FIGS. 10A, 10B and 10C are graphs of serum IgG antibody
levels to NTHi outer membrane proteins D (A), P6 (B) and OMP26 (C)
in healthy children increases with age. Boxplots of the geometric
mean concentration (ng/mL) displayed as a bar, 25.Salinity. and
75.Salinity. of the data displayed as the lower and upper limit of
the box and the 95% confidence interval displayed as a short
horizontal dash of antibody in sera of children taken during 7
sampling visits at 6, 9, 12, 15, 18, 24 and 30 months of age. The
number of sera included at each time point was 100, 88, 60, 59, 52,
43, and 8. Boxplots of the geometric mean concentration (ng/ml)
displayed as a bar, 25% ile and 75% ile of the data displayed as
the lower and upper limit of the box and the 95% confidence
interval displayed as short horizontal dash of antibody in sera of
children taken during 7 sampling visits at 6, 9, 12, 15, 18, 24 and
30 months of age. The number of sera included at each time point
was 100, 88, 60, 59, 52, 43, and 8.
[0028] FIGS. 11A, 11B and 11C show graphs comparing serum IgG
antibody levels to NTHi outer membrane proteins protein D (A), P6
(B) and OMP 26 (C) in NP colonized (.quadrature.NTHi+) and
non-colonized ( NTHi-) healthy children from 6 months to 24 months
of age. * indicates that the differences in colonized vs.
non-colonized children were significant for protein D at visit 15,
18 and 24-30 months with p value is equal to 0.04, 0.01 and 0.02
respectively and for P6 at 6, 9 and 15 months with p value 0.0003,
0.02 and 0.003 respectively.
[0029] FIG. 12 is a comparison of convalescent serum IgG levels to
NTHi outer membrane proteins D, P6 and OMP26 following an AOM or an
NP colonization event. X-axis represents the number of cumulative
data points. o=individual IgG level following an AOM event;
x=individual IgG level following and NP colonization event.
[0030] FIG. 13 shows graphs of IgG, IgM and IgA antibody levels
comparison to NTHi outer membrane proteins D, P6 and OMP26 in acute
(top) and convalescent (bottom) sera of 9 children with NTHi AOM.
Antibodies concentrations were summarized as geometric mean
concentration with 95% confidence intervals.
[0031] FIGS. 14A, 14B and 14C are graphs of individual IgG, IgM and
IgA antibody levels to NTHi outer membrane proteins D (A), P6 (B)
and OMP26 (C) in acute and convalescent sera of 9 children with
NTHi AOM.
[0032] FIG. 15 is the gating strategy for enumerating cytokine
specific memory CD4.sup.+ T-cells among children. To exclude cell
debris and clumps, cells were first gated based on their forward-
and side-scatter properties followed by sequential gating on
CD4.sup.+ CD45RA.sup.Low T-cells and then to CD3.sup.+CD69.sup.+
cytokine positive cells before gating on to TNF-.alpha. vs. other
cytokines. Low frequency responders were confirmed by excessive
back gating. Preliminarily, whole assay was standardized and
compared to multiplex bead array (CBA, BD Biosciences) for the
detection of CD4.sup.+ T-cell cytokine profiles.
[0033] FIGS. 16A and 16B are graphs of memory T cells. [A] Percent
frequencies of memory CD4.sup.+ T-cell subsets producing various
cytokines (IFN-.gamma., IL-4, IL-2 & IL-17a) against six
pneumococcal antigens in the circulation of non-otitis prone and
otitis prone children while un-stimulated control serve as a
negative control. Bar graphs represent normalized mean percentage
values of CD69.sup.+ CD4.sup.+ T-cells gated on CD45RA.sup.Low,
following antigen stimulations. Absolute blood counts were
calculated for the cytokine producing cells in case of PhtD
antigen. Error bars represent SEM; P values were calculated using
Mann Whitney test. *P<0.05; **P<0.005. [B] PBMC samples from
non otitis-prone and otitis-prone children were stimulated with SEB
and cytokine production was observed in CD45RA.sup.Low CD4.sup.+
T-cell population (p>0.5).
[0034] FIGS. 17A and 17B are graphs of IgG to different antigens.
[A] Comparison of IgG responses to five pneumococcal protein
antigens (PhtD, LytB, PcpA, PhtE and PlyD1) in the serum samples of
two cohorts of non-otitis prone and otitis prone children.
*P<0.05; **P<0.005; ***P<0.0005. Y-axis represents
Geometric mean titers and error bars are upper 95% confidence
intervals. [B] IgG responses to NTHi protein antigens (P6, OMP26
and Protein D) were also observed in the serum samples of two
cohorts of non-otitis prone and otitis prone children. *P<0.05.
Y-axis represents Geometric mean titers and error bars are upper
95% confidence intervals.
[0035] FIGS. 18A, 18B and 18 C are graphs of memory B cells and
IgG. FIG. 18A represents frequencies of antigen-specific memory B
cells enumerated in the same cohorts (mean.+-.SEM). Bar graphs
shows mean.+-.SEM of serum IgG titers to 5-pneumococcal protein
antigens in non-otitis prone (n=12) and otitis prone children
(n=10) (B). A correlation of PhtD specific serum antibody titers to
the PhtD specific percentages of antigen specific memory B cells is
shown in otitis prone (empty circles) and non-otitis prone children
(filled circles) (C). P values were calculated using Mann Whitney
test. *P<0.05 **<0.005.
DETAILED DESCRIPTION OF THE INVENTION
A. Compositions
1. Acute Otitis Media (AOM)
[0036] Otitis media is an inflammation of the middle ear, the space
behind the ear drum. It is one of the two conditions that are
commonly thought of as ear infections, the other being Otitis
externa. Inflammation of the middle ear in otitis media is caused
by a bacterial or viral infection. Ear infections are very common
in childhood, and include acute and recurrent- or
chronic-conditions; all of which involve inflammation of the ear
drum (tympanic membrane), and are usually associated with a buildup
of fluid in the space behind the ear drum (middle ear space). This
inflammation and fluid buildup results in pain which causes
caregivers or patients to readily seek medical attention for the
condition.
[0037] Inflammation in the middle ear space, and the associated
pain, are the essence of all otitis media infections. Once the
middle ear space is filled with fluid, hearing will be dampened
(conductive hearing impairment) until the condition improves. In
many individuals, for the reasons discussed below, the condition is
recurrent and will happen several times in a lifetime (chronic or
recurrent otitis media).
[0038] There are essentially two types of otitis media recognized,
each with a separate diagnosis and separate causes and general
symptomatic profiles. Otitis Media with Effusion (OME) is primarily
a non specific inflammatory response characterized by fluid behind
the ear. Acute Otis Media (AOM) with effusion is an infectious
disease characterized by rapid onset, pain, inflammation and the
like of the middle ear. It appears that 40 to 50% of AOM in young
children is caused by Streptococcus pneumonia, 20 to 30% by
Haemophilus influenza and 10 to 15% by Moraxella catarrhalis.
Recurrent AOM has been associated with excessive levels of S.
pneumonia and Haemophilus influenzae. Additionally, low levels of
antioxidants, including glutathione in particular, have also been
shown to correlate with recurrent AOM (Cemek et al., International
Journal of Pediatric Otorhinolaryngology, Volume 69, Issue 6, Pages
823-827).
[0039] Treatment of AOM has classically been accomplished with
antibacterial medications. Classic antibacterial treatment based on
the particular organism and its susceptibility to the antibiotic
rather than the disease state has been the mainstay of most
bacterial type infections. U.S. Pat. No. 6,987,093 discloses the
use of Azithromycin to treat AOM. Of course a problem with
antibiotic use is the eventual resistance of the particular
antibiotic to the strains that cause AOM. It is clear that many
high dosage antimicrobial treatments have a number of untoward side
effects.
i. Haemophilus influenza a. Outer Membrane Protein (OMP) P6
[0040] The recombinant outer membrane protein of the invention may
be derived from any NTHi bacterial outer membrane.
[0041] P6 is an outer membrane protein found on Nontypable H.
influenzae (NTHi) which is the major cause of acute otitis media
(AOM) (Casey et al. Pediatr Infect Dis J. 29:304-09, 2010). P6 was
identified as highly specific marker for NTHi (Murphy et al. J
Infect Dis. 152:1300-07, 1985). P6 is highly conserved among NTHi
strains (Nelson et al. Infect Immun 59:2658-63, 1991). Since P6 is
a surface protein, it is the target of human serum bactericidal
antibodies (Bogdan et al. Infect Immun 63:4395-4401, 1995; Murphy
et al. J Clin Invest 78:1020-27, 1986; De Maria et al. Infect Immun
64:5187-92, 1996).
[0042] P6 has previously been defined by its structure via NMR
spectroscopy (Orban et al. Biochemistry 45:2122-28, 2006). The
structure is hereby incorporated by reference. P6 is further
defined in EP 281673 (State University of New York) which is hereby
incorporated by reference.
The protein sequence for Haemophilus influenzae P6 Amino Acid
sequence (SEQ ID NO:1):
TABLE-US-00001 1 mnkfvksllv agsvaalaac sssnndaagn gaaqtfggys
vadlqqrynt vyfgfdkydi 61 tgeyvqilda haaylnatpa akvlvegntd
ergtpeynia lgqrradavk gylagkgvda 121 gklgtvsyge ekpavlghde
aaysknrrav lay
One letter coded Amino Acid sequence (SEQ ID NO:1):
TABLE-US-00002 MNKFVKSLLVAGSVAALAACSSSNNDAAGNGAAQTFGGYSVADLQQRYN
TVYFGFDKYDITGEYVQILDAHAAYLNATPAAKVLVEGNTDERGTPEYN
IALGQRRADAVKGYLAGKGVDAGKLGTVSYGEEKPAVLGHDEAAYSKNR RAVLAY
b. Protein D
[0043] Information about protein D can be found in EP 594610 by
Glaxo Smith Kline, which is herein incorporated by Reference in its
entirety at least for information related to protein D and
NTHi.
2. Vaccines
[0044] One composition disclosed herein is a vaccine. The vaccine
can contain nucleic acids, amino acids or a combination thereof. A
vaccine (or an immunogenic composition) comprises an immunogenic
amount (preferably an effective or protective amount) of a
composition, such as an outer membrane protein, (either isolated or
purified, or present in an outer membrane vesicle, ghost or killed,
live, or live-attenuated whole cell preparation) in a
pharmaceutically acceptable excipient, and an optional adjuvant. In
this context, immunogenic amount can be defined as a sufficient
quantity of protein to elicit an antibody response in a host.
[0045] An immunogenic amount of one of the disclosed compositions
can be formulated in a pharmaceutically acceptable excipient, and
an optional adjuvant, to prevent or treat Haemophilus influenzae
disease (preferably otitis media, sinusitis, conjunctivitis, or
lower respiratory tract infection). Vaccines can be used to induce
an immune response in a mammal susceptible to Haemophilus
influenzae infection by administering to the mammal an effective
amount of the vaccine (an effective amount being an amount capable
of protecting a host to some degree against an NTHi infection). A
vaccine can also prevent Haemophilus influenzae infection by
administration to a mammal in an effective amount.
[0046] Vaccines are capable of eliciting a cross-protective immune
response against a large variety of NTHi strains (particularly
where one or more modified loops are integrated into an NTHi outer
membrane protein).
[0047] A preferred vaccine comprises a recombinant NTHi outer
membrane protein, preferably P6, as such vaccines can effectively
protect a host against otitis media by immunization with a single
molecule.
[0048] Vaccines can elicit a humoral response, cell-mediated immune
response or a combination thereof. Ideally, the immune response
provides protection upon subsequent challenge with NTHi. However,
protective immunity is not required.
[0049] Additionally, the proteins of the present invention are
preferably adjuvanted in the vaccine formulation of the invention.
Suitable adjuvants include an aluminium salt such as aluminium
hydroxide gel (alum) or aluminium phosphate, but may also be a salt
of calcium, iron or zinc, or may be an insoluble suspension of
acylated tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes. Other known
adjuvants include CpG containing oligonucleotides. The
oligonucleotides are characterized in that the CpG dinucleotide is
unmethylated. Such oligonucleotides are well known and are
described in, for example WO96/02555.
[0050] Further preferred adjuvants are those which induce an immune
response preferentially of the TH1 type. High levels of Th1-type
cytokines tend to favor the induction of cell mediated immune
responses to the given antigen, whilst high levels of Th2-type
cytokines tend to favor the induction of humoral immune responses
to the antigen. Suitable adjuvant systems include, for example
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A (3D-MPL), or a combination of 3D-MPL together with an
aluminium salt. CpG oligonucleotides also preferentially induce a
TH1 response. An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96/33739. A particularly potent
adjuvant formulation involving QS21 3D-MPL & tocopherol in an
oil in water emulsion is described in WO 95/17210 and is a
preferred formulation.
3. Detection Label
[0051] The disclosed compositions can comprise a detection label,
also referred to as detectable agent. A variety of detectable
agents are useful in the disclosed methods. As used herein, the
term "detectable agent" refers to any molecule which can be
detected. Useful detectable agents include compounds and molecules
that can be administered in vivo and subsequently detected.
Detectable agents useful in the disclosed compositions and methods
include yet are not limited to radiolabels and fluorescent
molecules. The detectable agent can be, for example, any molecule
that facilitates detection, either directly or indirectly,
preferably by a non-invasive and/or in vivo visualization
technique. For example, a detectable agent can be detectable by any
known imaging techniques, including, for example, a radiological
technique, a magnetic resonance technique, or an ultrasound
technique. Detectable agents can include, for example, a
contrasting agent, e.g., where the contrasting agent is ionic or
non-ionic. In some embodiments, for instance, the detectable agent
comprises a tantalum compound and/or a barium compound, e.g.,
barium sulfate. In some embodiments, the detectable agent comprises
iodine, such as radioactive iodine. In some embodiments, for
instance, the detectable agent comprises an organic iodo acid, such
as iodo carboxylic acid, triiodophenol, iodoform, and/or
tetraiodoethylene. In some embodiments, the detectable agent
comprises a non-radioactive detectable agent, e.g., a
non-radioactive isotope. For example, Gd can be used as a
non-radioactive detectable agent in certain embodiments.
[0052] Other examples of detectable agents include molecules which
emit or can be caused to emit detectable radiation (e.g.,
fluorescence excitation, radioactive decay, spin resonance
excitation, etc.), molecules which affect local electromagnetic
fields (e.g., magnetic, ferromagnetic, ferromagnetic, paramagnetic,
and/or superparamagnetic species), molecules which absorb or
scatter radiation energy (e.g., chromophores and/or fluorophores),
quantum dots, heavy elements and/or compounds thereof. See, e.g.,
detectable agents described in U.S. Publication No. 2004/0009122.
Other examples of detectable agents include a proton-emitting
molecules, a radiopaque molecules, and/or a radioactive molecules,
such as a radionuclide like Tc-99m and/or Xe-13. Such molecules can
be used as a radiopharmaceutical. In still other embodiments, the
disclosed compositions can comprise one or more different types of
detectable agents, including any combination of the detectable
agents disclosed herein.
[0053] Useful fluorescent agents include fluorescein isothiocyanate
(FITC), 5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin,
BODIPY.RTM., Cascade Blue.RTM., Oregon Green.RTM., pyrene,
lissamine, xanthenes, acridines, oxazines, phycoerythrin,
macrocyclic chelates of lanthanide ions such as Quantum Dye.TM.,
fluorescent energy transfer dyes, such as thiazole orange-ethidium
heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Examples of other specific fluorescent labels include
3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine
(5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red,
Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon
Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon
Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G,
BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate,
Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy F1,
Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor
RW Solution, Calcofluor White, Calcophor White ABT Solution,
Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,
Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin,
CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic
Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH--CH3,
Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid,
Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF,
Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced
Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2,
Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl
Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue,
Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF,
Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),
Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue,
Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF,
MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine,
Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear
Yellow, Nylosan Brilliant Flavin EBG, Oxadiazole, Pacific Blue,
Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL,
Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,
Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin,
Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant
Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD,
Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,
Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron
Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B,
Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene
Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can
C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R,
Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol
Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC,
Xylene Orange, and XRITC.
[0054] Particularly useful fluorescent labels include fluorescein
(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine
(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. The absorption and emission maxima, respectively,
for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),
Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703
nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous
detection. Other examples of fluorescein dyes include
6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein
(TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused
phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
Fluorescent labels can be obtained from a variety of commercial
sources, including Amersham Pharmacia Biotech, Piscataway, N.J.;
Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,
Ohio. Fluorescent probes and there use are also described in
Handbook of Fluorescent Probes and Research Products by Richard P.
Haugland.
[0055] Further examples of radioactive detectable agents include
gamma emitters, e.g., the gamma emitters In-111, I-125 and I-131,
Rhenium-186 and 188, and Br-77 (see. e.g., Thakur, M. L. et al.,
Throm Res. Vol. 9 pg. 345 (1976); Powers et al., Neurology Vol. 32
pg. 938 (1982); and U.S. Pat. No. 5,011,686); positron emitters,
such as Cu-64, C-11, and O-15, as well as Co-57, Cu-67, Ga-67,
Ga-68, Ru-97, Tc-99m, In-113m, Hg-197, Au-198, and Pb-203. Other
radioactive detectable agents can include, for example tritium,
C-14 and/or thallium, as well as Rh-105, I-123, Nd-147, Pm-151,
Sm-153, Gd-159, Tb-161, Er-171 and/or Tl-201.
[0056] The use of Technitium-99m (Tc-99m) is preferable and has
been described in other applications, for example, see U.S. Pat.
No. 4,418,052 and U.S. Pat. No. 5,024,829. Tc-99m is a gamma
emitter with single photon energy of 140 keV and a half-life of
about 6 hours, and can readily be obtained from a Mo-99/Tc-99
generator.
[0057] In some embodiments, compositions comprising a radioactive
detectable agent can be prepared by coupling a targeting moiety
with radioisotopes suitable for detection. Coupling can occur via a
chelating agent such as diethylenetriaminepentaacetic acid (DTPA),
4,7,10-tetraazacyclododecane-N--,N',N'',N'''-tetraacetic acid
(DOTA) and/or metallothionein, any of which can be covalently
attached to the targeting moiety. In some embodiments, an aqueous
mixture of technetium-99m, a reducing agent, and a water-soluble
ligand can be prepared and then allowed to react with a disclosed
targeting moiety. Such methods are known in the art, see e.g.,
International Publication No. WO 99/64446. In some embodiments,
compositions comprising radioactive iodine, can be prepared using
an exchange reaction. For example, exchange of hot iodine for cold
iodine is well known in the art. Alternatively, a radio-iodine
labeled compound can be prepared from the corresponding bromo
compound via a tributylstannyl intermediate.
[0058] Magnetic detectable agents include paramagnetic contrasting
agents, e.g., gadolinium diethylenetriaminepentaacetic acid, e.g.,
used with magnetic resonance imaging (MRI) (see, e.g., De Roos, A.
et al., Int. J. Card. Imaging Vol. 7 pg. 133 (1991)). Some
preferred embodiments use as the detectable agent paramagnetic
atoms that are divalent or trivalent ions of elements with an
atomic number 21, 22, 23, 24, 25, 26, 27, 28, 29, 42, 44, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. Suitable ions
include, but are not limited to, chromium(III), manganese(II),
iron(II), iron(III), cobalt(II), nickel(II), copper(II),
praseodymium(III), neodymium(III), samarium(III) and
ytterbium(III), as well as gadolinium(III), terbiurn(III),
dysoprosium(III), holmium(III), and erbium(III). Some preferred
embodiments use atoms with strong magnetic moments, e.g.,
gadolinium(III).
[0059] In some embodiments, compositions comprising magnetic
detectable agents can be prepared by coupling a targeting moiety
with a paramagnetic atom. For example, the metal oxide or a metal
salt, such as a nitrate, chloride or sulfate salt, of a suitable
paramagnetic atom can be dissolved or suspended in a water/alcohol
medium, such as methyl, ethyl, and/or isopropyl alcohol. The
mixture can be added to a solution of an equimolar amount of the
targeting moiety in a similar water/alcohol medium and stirred. The
mixture can be heated moderately until the reaction is complete or
nearly complete. Insoluble compositions formed can be obtained by
filtering, while soluble compositions can be obtained by
evaporating the solvent. If acid groups on the chelating moieties
remain in the disclosed compositions, inorganic bases (e.g.,
hydroxides, carbonates and/or bicarbonates of sodium, potassium
and/or lithium), organic bases, and/or basic amino acids can be
used to neutralize acidic groups, e.g., to facilitate isolation or
purification of the composition.
[0060] The detectable agent can be coupled to the composition in
such a way so as not to interfere with the ability of the vaccine
to generate an immune response. The detectable agent can be
directly or indirectly bound or conjugated to the disclosed
compositions.
4. Therapeutic Agents
[0061] As used herein, the term "therapeutic agent" means a
molecule which can have one or more biological activities in a
normal or pathologic tissue. A variety of therapeutic agents can be
used. The therapeutic agent can comprise a compound or composition
for treating viral, bacterial or fungal diseases. The therapeutic
agent can comprise a compound or composition to
[0062] A therapeutic agent can be a therapeutic polypeptide. As
used herein, a therapeutic polypeptide can be any polypeptide with
a biologically useful function. Useful therapeutic polypeptides
encompass, without limitation, cytokines, antibodies, cytotoxic
polypeptides; and pro-apoptotic polypeptides. As non-limiting
examples, useful therapeutic polypeptides can be a cytokine such as
tumor necrosis factor-.alpha. (TNF-.alpha.), tumor necrosis
factor-.beta. (TNF-.beta.), granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), interferon-.alpha. (IFN-.alpha.); interferon-.gamma.
(IFN-.gamma.), interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6),
interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12
(IL-12), lymphotactin (LTN) or dendritic cell chemokine 1 (DC-CK1);
a cytotoxic polypeptide including a toxin or caspase, for example,
diphtheria toxin A chain, Pseudomonas exotoxin A, cholera toxin, a
ligand fusion toxin such as DAB389EGF or ricin; or one of those
described further herein or known in the art (see below). It is
understood that these and other polypeptides with biological
activity can be a "therapeutic polypeptide."
[0063] The compositions disclosed herein can also be used at a site
of inflammation or injury. Agents useful for this purpose can
include therapeutic agents belonging to several basic groups
including anti-inflammatory agents which prevent inflammation,
restenosis preventing drugs which prevent tissue growth,
anti-thrombogenic drugs which inhibit or control formation of
thrombus or thrombolytics, and bioactive agents which regulate
tissue growth and enhance healing of the tissue. Examples of useful
therapeutic agents include but are not limited to steroids,
fibronectin, anti-clotting drugs, anti-platelet function drugs,
drugs which prevent smooth muscle cell growth on inner surface wall
of vessel, heparin, heparin fragments, aspirin, coumadin, tissue
plasminogen activator (TPA), urokinase, hirudin, streptokinase,
antiproliferatives (methotrexate, cisplatin, fluorouracil,
Adriamycin), antioxidants (ascorbic acid, beta carotene, vitamin
E), antimetabolites, thromboxane inhibitors, non-steroidal and
steroidal anti-inflammatory drugs, beta and calcium channel
blockers, genetic materials including DNA and RNA fragments,
complete expression genes, antibodies, lymphokines, growth factors,
prostaglandins, leukotrienes, laminin, elastin, collagen, and
integrins.
[0064] Useful therapeutic agents also can be antimicrobial
peptides. For example, disclosed are agents comprising an
antimicrobial peptide, where the composition is selectively
internalized and exhibits a high toxicity to the targeted area.
Useful antimicrobial peptides can have low mammalian cell toxicity
when not incorporated into the composition. As used herein, the
term "antimicrobial peptide" means a naturally occurring or
synthetic peptide having antimicrobial activity, which is the
ability to kill or slow the growth of one or more microbes. An
antimicrobial peptide can, for example, kill or slow the growth of
one or more strains of bacteria including Gram-positive or
Gram-negative bacteria, or fungi or protozoa. Thus, an
antimicrobial peptide can have, for example, bacteriostatic or
bacteriocidal activity against, for example, one or more strains of
Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus.
While not wishing to be bound by the following, an antimicrobial
peptide can have biological activity due to the ability to form ion
channels through membrane bilayers as a consequence of
self-aggregation.
[0065] An antimicrobial peptide is typically highly basic and can
have a linear or cyclic structure. As discussed further below, an
antimicrobial peptide can have an amphipathic .alpha.-helical
structure (see U.S. Pat. No. 5,789,542; Javadpour et al., J. Med.
Chem. 39:3107-3113 (1996); and Blondelle and Houghten, Biochem. 31:
12688-12694 (1992)). An antimicrobial peptide also can be, for
example, a .beta.-strand/sheet-forming peptide as described in
Mancheno et al., J. Peptide Res. 51:142-148 (1998).
[0066] An antimicrobial peptide can be a naturally occurring or
synthetic peptide. Naturally occurring antimicrobial peptides have
been isolated from biological sources such as bacteria, insects,
amphibians, and mammals and are thought to represent inducible
defense proteins that can protect the host organism from bacterial
infection. Naturally occurring antimicrobial peptides include the
gramicidins, magainins, mellitins, defensins and cecropins (see,
for example, Maloy and Kari, Biopolymers 37:105-122 (1995);
Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994); Bessalle et
al., FEBS 274:-151-155 (1990.); and Blondelle and Houghten in
Bristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168
Academic Press, San Diego). An antimicrobial peptide also can be an
analog of a natural peptide, especially one that retains or
enhances amphipathicity (see below).
[0067] An antimicrobial peptide incorporated into the composition
disclosed herein can have low mammalian cell toxicity when linked
to the composition. Mammalian cell toxicity readily can be assessed
using routine assays. As an example, mammalian cell toxicity can be
assayed by lysis of human erythrocytes in vitro as described in
Javadpour et al., supra, 1996. An antimicrobial peptide having low
mammalian cell toxicity is not lytic to human erythrocytes or
requires concentrations of greater than 100 .mu.M for lytic
activity, preferably concentrations greater than 200, 300, 500 or
1000 .mu.M.
[0068] In one embodiment, disclosed are compositions in which the
antimicrobial peptide portion promotes disruption of mitochondrial
membranes when internalized by eukaryotic cells. In particular,
such an antimicrobial peptide preferentially disrupts mitochondrial
membranes as compared to eukaryotic membranes. Mitochondrial
membranes, like bacterial membranes but in contrast to eukaryotic
plasma membranes, have a high content of negatively charged
phospholipids. An antimicrobial peptide can be assayed for activity
in disrupting mitochondrial membranes using, for example, an assay
for mitochondrial swelling or another assay well known in the
art.
[0069] An antimicrobial peptide that induces significant
mitochondrial swelling at, for example, 50 .mu.M, 40 .mu.M, 30
.mu.M, 20 .mu.M, 10 .mu.M, or less, is considered a peptide that
promotes disruption of mitochondrial membranes.
[0070] Antimicrobial peptides generally have random coil
conformations in dilute aqueous solutions, yet high levels of
helicity can be induced by helix-promoting solvents and amphipathic
media such as micelles, synthetic bilayers or cell membranes.
.alpha.-Helical structures are well known in the art, with an ideal
.alpha.-helix characterized by having 3.6 residues per turn and a
translation of 1.5 .ANG. per residue (5.4 .ANG. per turn; see
Creighton, Proteins: Structures and Molecular Properties W.H
Freeman, New York (1984)). In an amphipathic .alpha.-helical
structure, polar and non-polar amino acid residues are aligned into
an amphipathic helix, which is an .alpha.-helix in which the
hydrophobic amino acid residues are predominantly on one face, with
hydrophilic residues predominantly on the opposite face when the
peptide is viewed along the helical axis.
[0071] Antimicrobial peptides of widely varying sequence have been
isolated, sharing an amphipathic .alpha.-helical structure as a
common feature (Saberwal et al., Biochim. Biophys. Acta
1197:109-131 (1994)). Analogs of native peptides with amino acid
substitutions predicted to enhance amphipathicity and helicity
typically have increased antimicrobial activity. In general,
analogs with increased antimicrobial activity also have increased
cytotoxicity against mammalian cells (Maloy et al., Biopolymers
37:105-122 (1995)).
[0072] As used herein in reference to an antimicrobial peptide, the
term "amphipathic .alpha.-helical structure" means an .alpha.-helix
with a hydrophilic face containing several polar residues at
physiological pH and a hydrophobic face containing nonpolar
residues. A polar residue can be, for example, a lysine or arginine
residue, while a nonpolar residue can be, for example, a leucine or
alanine residue. An antimicrobial peptide having an amphipathic
.alpha.-helical structure generally has an equivalent number of
polar and nonpolar residues within the amphipathic domain and a
sufficient number of basic residues to give the peptide an overall
positive charge at neutral pH (Saberwal et al., Biochim. Biophys.
Acta 1197:109-131 (1994)). One skilled in the art understands that
helix-promoting amino acids such as leucine and alanine can be
advantageously included in an antimicrobial peptide (see, for
example, Creighton, supra, 1984). Synthetic, antimicrobial peptides
having an amphipathic .alpha.-helical structure are known in the
art, for example, as described in U.S. Pat. No. 5,789,542 to
McLaughlin and Becker.
[0073] Anti-fungal agents that can be administered with the
compounds of the invention include, but are not limited to, polyene
antifungals (e.g., amphotericin and nystatin), azole antifungals
(e.g., ketoconazole, miconazole, fluconazole, itraconazole,
posaconazole, ravuconazole, voriconazole, clotrimazole, econazole,
oxiconazole, sulconazole, terconazole, butoconazole, isavuconazole,
and tioconazole), amorolfine, butenafine, naftifine, terbinafine,
flucytosine, nikkomycin Z, echinocandins (e.g., caspofungin,
micafungin (FK463), anidulafungin (LY303366)), griseofulvin,
ciclopiroxolamine, tolnaftate, intrathecal, 5-fluorocytosine,
MK0991 (Merck), haloprogrin, and undecylenate.
[0074] Anti-bacterial agents that can be administered with the
compounds of the invention include, but are not limited to, sulfa
drugs (e.g., sulfanilamide), folic acid analogs (e.g.,
trimethoprim), beta-lactams (e.g., penacillin, cephalosporins),
aminoglycosides (e.g., stretomycin, kanamycin, neomycin,
gentamycin), tetracyclines (e.g., chlorotetracycline,
oxytetracycline, and doxycycline), macrolides (e.g., erythromycin,
azithromycin, and clarithromycin), lincosamides (e.g.,
clindamycin), streptogramins (e.g., quinupristin and dalfopristin),
fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and
moxifloxacin), polypeptides (e.g., polymixins), rifampin,
mupirocin, cycloserine, aminocyclitol (e.g., spectinomycin),
glycopeptides (e.g., vancomycin), oxazolidinones (e.g., linezolid),
ribosomes, chloramphenicol, fusidic acid, and metronidazole.
[0075] Anti-viral agents that can be administered with the
compounds of the invention include, but are not limited to,
Emtricitabine (FTC); Lamivudine (3TC); Carbovir; Acyclovir;
Interferon; Famciclovir; Penciclovir; Zidovudine (AZT); Didanosine
(ddI); Zalcitabine (ddC); Stavudine (d4T); Tenofovir DF (Viread);
Abacavir (ABC); L-(-)-FMAU; L-DDA phosphate prodrugs;
.beta.-D-dioxolane nucleosides such as .beta.-D-dioxolanyl-guanine
(DG), .beta.-D-dioxolanyl-2,6-diaminopurine (DAPD), and
.beta.-D-dioxolanyl-6-chloropurine (ACP); non-nucleoside RT
inhibitors such as Nevirapine (Viramune), MKC-442, Efavirenz
(Sustiva), Delavirdine (Rescriptor); protease inhibitors such as
Amprenavir, Atazanavir, Fosamprenavir, Indinavir, Kaletra,
Nelfinavir, Ritonavir, Saquinavir, AZT, DMP-450; combination
treatments such as Epzicom (ABC+3TC), Trizivir (ABC+3TC+AZT),
Truvada (FTC+Viread); Omega IFN (BioMedicines Inc.); BILN-2061
(Boehringer Ingelheim); Summetrel (Endo Pharmaceuticals Holdings
Inc.); Roferon A (F. Hoffman-La Roche); Pegasys (F. Hoffman-La
Roche); Pegasys/Ribaravin (F. Hoffman-La Roche); CellCept (F.
Hoffman-La Roche); Wellferon (GlaxoSmithKline); Albuferon-alpha
(Human Genome Sciences Inc.); Levovirin (ICN Pharmaceuticals);
IDN-6556 (Idun Pharmaceuticals); IP-501 (Indevus Pharmaceuticals);
Actimmune (InterMune Inc.); Infergen A (InterMune Inc.); ISIS 14803
(ISIS Pharamceuticals Inc.); JTK-003 (Japan Tobacco Inc.);
Pegasys/Ceplene (Maxim Pharmaceuticals); Ceplene (Maxim
Pharmaceuticals); Civacir (Nabi Biopharmaceuticals Inc.); Intron
A/Zadaxin (RegeneRx); Levovirin (Ribapharm Inc.); Viramidine
(Ribapharm Inc.); Heptazyme (Ribozyme Pharmaceuticals); Intron A
(Schering-Plough); PEG-Intron (Schering-Plough); Rebetron (Schering
Plough); Ribavirin (Schering-Plough); PEG-Intron/Ribavirin
(Schering-Plough); Zadazim (SciClone); Rebif (Serono);
IFN-.beta./EMZ701 (Transition Therapeutics); T67 (Tularik Inc.);
VX-497 (Vertex Pharmaceuticals Inc.); VX-950/LY-570310 (Vertex
Pharmaceuticals Inc.); Omniferon (Viragen Inc.); XTL-002 (XTL
Biopharmaceuticals); SCH 503034 (Schering-Plough); isatoribine and
its prodrugs ANA971 and ANA975 (Anadys); R1479 (Roche Biosciences);
Valopicitabine (Idenix); NIM811 (Novartis); Actilon (Coley
Pharmaceuticals); Pradefovir (Metabasis. Therapeutics); zanamivir;
adefovir, adefovir dipivoxil, oseltamivir; vidarabine; gancyclovir;
valganciclovir; amantadine; rimantadine; relenza; tamiflu;
amantadine; entecavir; and pleconaril.
[0076] Anti-parasitic agents that can be administered with the
compounds of the invention include, but are not limited to,
avermectins, milbemycins, lufenuron, imidacloprid,
organophosphates, pyrethroids, sufanamides, iodquinol, diloxanide
furoate, metronidazole, paromycin, azithromycin, quinacrine,
furazolidone, tinidazole, ornidazole, bovine, colostrum, bovine
dialyzable leukocyte extract, chloroquine, chloroquine phosphate,
diclazuril, eflornithine, paromomycin, pentamidine, pyrimethamine,
spiramycin, trimethoprim-sulfamethoxazole, albendazole, quinine,
quinidine, tetracycline, pyrimethamine-sulfadoxine, mefloquine,
doxycycline, proguanil, clindamycin, suramin, melarsoprol,
diminazene, nifurtimox, spiroarsoranes, ketoconazole, terbinafine,
lovastatin, sodium stibobgluconate, N-methylglucamine antimonate,
amphotericin B, allopurinol, itraconazole, sulfadiazine, dapsone,
trimetrexate, clarithromycin, roxithromycin, atovaquone, aprinocid,
timidazole, mepacrine hydrochloride, emetine, polyaminopropyl
biguanide, paromomycin, benzimidazole, praziquantel, or
albendazole.
5. Peptides and Peptide Variants
[0077] In some forms, the compositions can be or include a peptide,
peptidomimetic, and/or amino acid segment. Unless the context
indicates otherwise, reference herein to "peptide" is intended to
refer also to amino acid segments, which can form a part of, or
constitute an entire, peptide. The disclosed peptides can be in
isolated form. As used herein in reference to the disclosed
peptides, the term "isolated" means a peptide that is in a form
that is relatively free from material such as contaminating
polypeptides, lipids, nucleic acids and other cellular material
that normally is associated with the peptide in a cell or that is
associated with the peptide in a library or in a crude
preparation.
[0078] The disclosed peptides and amino acid segments can have any
suitable length. The disclosed peptides can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed peptides also
can be useful in the context of a significantly longer sequence.
Thus, the peptides can have, for example, a length of up to 50,
100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In
particular embodiments, a peptide can have a length of at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further
embodiments, a peptide can have a length of 5 to 200 residues, 5 to
100 residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues,
5 to 60 residues, 5 to 50 residues, 5 to 40 residues, 5 to 30
residues, 5 to 20 residues, 5 to 15 residues, 5 to 10 residues, 10
to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80
residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues,
10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 20 to 200
residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues,
20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40
residues or 20 to 30 residues. As used herein, the term "residue"
refers to an amino acid or amino acid analog.
[0079] The disclosed amino acid segments can have, for example, a
relatively short length of less than six, seven, eight, nine, ten,
12, 15, 20, 25, 30, 35 or 40 residues. The disclosed amino acid
segments also can be useful in the context of a significantly
longer sequence. Thus, the amino acid segments can have, for
example, a length of up to 50, 100, 150, 200, 250, 300, 400, 500,
1000 or 2000 residues. In particular embodiments, an amino acid
segment can have a length of at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 or 200 residues. In further embodiments, an amino acid
segment can have a length of 5 to 200 residues, 5 to 100 residues,
5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60
residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5
to 20 residues, 5 to 15 residues, 5 to 10 residues, 10 to 200
residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues,
10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40
residues, 10 to 30 residues, 10 to 20 residues, 20 to 200 residues,
20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70
residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues
or 20 to 30 residues. As used herein, the term "residue" refers to
an amino acid or amino acid analog.
[0080] As this specification discusses various proteins, protein
sequences, peptides, peptides sequences, and amino acid sequences,
it is understood that the nucleic acids that can encode those
sequences are also disclosed. This would include all degenerate
sequences related to a specific protein sequence, i.e. all nucleic
acids having a sequence that encodes one particular protein
sequence as well as all nucleic acids, including degenerate nucleic
acids, encoding the disclosed variants and derivatives of the
protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. The disclosed peptides and
proteins can be coupled to each other via peptide bonds to form
fusion peptides and proteins.
[0081] The disclosed peptides and amino acid segments can be
modified. As used herein, a "methylated derivative" of a protein,
peptide, amino acid segment, amino acid sequence, etc. refers to a
form of the protein, peptide, amino acid segment, amino acid
sequence, etc. that is methylated. Unless the context indicates
otherwise, reference to a methylated derivative of a protein,
peptide, amino acid segment, amino acid sequence, etc. does no
include any modification to the base protein, peptide, amino acid
segment, amino acid sequence, etc. other than methylation.
Methylated derivatives can also have other modifications, but such
modifications generally will be noted. For example, conservative
variants of an amino acid sequence would include conservative amino
acid substitutions of the based amino acid sequence. Thus,
reference to, for example, a "methylated derivative" of a specific
amino acid sequence "and conservative variants thereof" would
include methylated forms of the specific amino acid sequence and
methylated forms of the conservative variants of the specific amino
acid sequence, but not any other modifications of derivations. As
another example, reference to a methylated derivative of an amino
acid segment that includes amino acid substitutions would include
methylated forms of the amino acid sequence of the amino acid
segment and methylated forms of the amino acid sequence of the
amino acid segment include amino acid substitutions.
[0082] Protein variants and derivatives are well understood by
those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof can
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure.
[0083] As used herein in reference to a specified amino acid
sequence, a "conservative variant" is a sequence in which a first
amino acid is replaced by another amino acid or amino acid analog
having at least one biochemical property similar to that of the
first amino acid; similar properties include, for example, similar
size, charge, hydrophobicity or hydrogen-bonding capacity.
Conservative variants are also referred to herein as "conservative
amino acid substitutions," "conservative amino acid variants,"
"conservative substitutions," and similar phrase. A "conservative
derivative" of a reference sequence refers to an amino acid
sequence that differs from the reference sequences only in
conservative substitutions.
[0084] As an example, a conservative variant can be a sequence in
which a first uncharged polar amino acid is conservatively
substituted with a second (non-identical) uncharged polar amino
acid such as cysteine, serine, threonine, tyrosine, glycine,
glutamine or asparagine or an analog thereof. A conservative
variant also can be a sequence in which a first basic amino acid is
conservatively substituted with a second basic amino acid such as
arginine, lysine, histidine, 5-hydroxylysine, N-methyllysine or an
analog thereof. Similarly, a conservative variant can be a sequence
in which a first hydrophobic amino acid is conservatively
substituted with a second hydrophobic amino acid such as alanine,
valine, leucine, isoleucine, proline, methionine, phenylalanine or
tryptophan or an analog thereof. In the same way, a conservative
variant can be a sequence in which a first acidic amino acid is
conservatively substituted with a second acidic amino acid such as
aspartic acid or glutamic acid or an analog thereof; a sequence in
which an aromatic amino acid such as phenylalanine is
conservatively substituted with a second aromatic amino acid or
amino acid analog, for example, tyrosine; or a sequence in which a
first relatively small amino acid such as alanine is substituted
with a second relatively small amino acid or amino acid analog such
as glycine or valine or an analog thereof. For example, the
replacement of one amino acid residue with another that is
biologically and/or chemically similar is known to those skilled in
the art as a conservative substitution. For example, a conservative
substitution would be replacing one hydrophobic residue for
another, or one polar residue for another. The substitutions
include combinations such as, for example, Gly, Ala; Val, Ile, Leu;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such
conservatively substituted variations of each explicitly disclosed
sequence are included within the mosaic polypeptides provided
herein. It is understood that conservative variants of the
disclosed amino acid sequences can encompass sequences containing,
for example, one, two, three, four or more amino acid substitutions
relative to the reference sequence, and that such variants can
include naturally and non-naturally occurring amino acid
analogs.
[0085] Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Examples of such substitutions, referred to as conservative
substitutions, can generally be made in accordance with the
following Table 6.
TABLE-US-00003 TABLE 6 Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn, Lys Glu
Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met
Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0086] Substantial changes in function or immunological identity
can be made by selecting substitutions that are less conservative,
i.e., selecting residues that differ more significantly in their
effect on maintaining (a) the structure of the polypeptide backbone
in the area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the protein properties will be those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or
by) an electronegative residue, e.g., glutamyl or aspartyl; or (d)
a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine,
in this case, (e) by increasing the number of sites for sulfation
and/or glycosylation. These can be referred to a less conservative
variants.
[0087] Peptides can have a variety of modifications. Modifications
can be used to change or improve the properties of the peptides.
For example, the disclosed peptides can be N-methylated,
O-methylated, S-methylated, C-methylated, or a combination at one
or more amino acids.
[0088] The amino and/or carboxy termini of the disclosed peptides
can be modified. Amino terminus modifications include methylation
(e.g., --NHCH.sub.3 or --N(CH.sub.3).sub.2), acetylation (e.g.,
with acetic acid or a halogenated derivative thereof such as
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group,
or blocking the amino terminus with any blocking group containing a
carboxylate functionality defined by RCOO-- or sulfonyl
functionality defined by R--SO.sub.2--, where R is selected from
the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and
the like, and similar groups. One can also incorporate a desamino
acid at the N-terminus (so that there is no N-terminal amino group)
to decrease susceptibility to proteases or to restrict the
conformation of the peptide compound. In preferred embodiments, the
N-terminus is acetylated with acetic acid or acetic anhydride.
[0089] Carboxy terminus modifications include replacing the free
acid with a carboxamide group or forming a cyclic lactam at the
carboxy terminus to introduce structural constraints. One can also
cyclize the disclosed peptides, or incorporate a desamino or
descarboxy residue at the termini of the peptide, so that there is
no terminal amino or carboxyl group, to decrease susceptibility to
proteases or to restrict the conformation of the peptide.
C-terminal functional groups of the disclosed peptides include
amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,
hydroxy, and carboxy, and the lower ester derivatives thereof, and
the pharmaceutically acceptable salts thereof.
[0090] One can replace the naturally occurring side chains of the
genetically encoded amino acids (or the stereoisomeric D amino
acids) with other side chains, for instance with groups such as
alkyl, lower (C.sub.1-6) alkyl, cyclic 4-, 5-, 6-, to 7-membered
alkyl, amide, amide lower alkyl amide di(lower alkyl), lower
alkoxy, hydroxy, carboxy and the lower ester derivatives thereof,
and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular,
proline analogues in which the ring size of the proline residue is
changed from 5 members to 4, 6, or 7 members can be employed.
Cyclic groups can be saturated or unsaturated, and if unsaturated,
can be aromatic or non-aromatic. Heterocyclic groups preferably
contain one or more nitrogen, oxygen, and/or sulfur heteroatoms.
Examples of such groups include the furazanyl, furyl,
imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,
morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,
1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,
pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,
pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl),
pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These
heterocyclic groups can be substituted or unsubstituted. Where a
group is substituted, the substituent can be alkyl, alkoxy,
halogen, oxygen, or substituted or unsubstituted phenyl.
[0091] One can also readily modify peptides by phosphorylation, and
other methods [e.g., as described in Hruby, et al. (1990) Biochem
J. 268:249-262].
[0092] The disclosed peptides also serve as structural models for
non-peptidic compounds with similar biological activity. Those of
skill in the art recognize that a variety of techniques are
available for constructing compounds with the same or similar
desired biological activity as the lead peptide compound, but with
more favorable activity than the lead with respect to solubility,
stability, and susceptibility to hydrolysis and proteolysis [See,
Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252]. These
techniques include, but are not limited to, replacing the peptide
backbone with a backbone composed of phosphonates, amidates,
carbamates, sulfonamides, secondary amines, and N-methylamino
acids.
[0093] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH--(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CHH.sub.2SO-- (These and others can be found in Spatola, A.
F. in Chemistry and Biochemistry of Amino Acids, Peptides, and
Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267
(1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,
Peptide Backbone Modifications (general review); Morley, Trends
Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot
Res 14:177-185 (1979) (--CH.sub.2NH--, CH.sub.2CH.sub.2); Spatola
et al. Life Sci 38:1243-1249 (1986) (--CHH.sub.2--S); Hann J. Chem.
Soc Perkin Trans. 1307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2--); Szelke et al. European Appin, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as .beta.-alanine,
.gamma.-aminobutyric acid, and the like.
[0094] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
can be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, can be accomplished, for example, by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0095] Certain post-translational derivatizations can be the result
of the action of recombinant host cells on the expressed
polypeptide. Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0096] It is understood that one way to define the variants and
derivatives of the disclosed amino acids sequences, amino acid
segments, peptides, proteins, etc. herein is through defining the
variants and derivatives in terms of homology/identity to specific
known sequences. For example, specifically disclosed are variants
of these and other amino acids sequences, amino acid segments,
peptides, proteins, etc. herein disclosed which have at least, 70%
or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
Those of skill in the art readily understand how to determine the
homology of two proteins. For example, the homology can be
calculated after aligning the two sequences so that the homology is
at its highest level.
[0097] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
can be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0098] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0099] It is understood that the description of conservative
variants and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative variants.
[0100] As this specification discusses various amino acids
sequences, amino acid segment sequences, peptide sequences, protein
sequences, etc., it is understood that nucleic acids that can
encode those sequences are also disclosed. This would include all
degenerate sequences related to a specific amino acid sequence,
i.e. all nucleic acids having a sequence that encodes one
particular amino acid sequence as well as all nucleic acids,
including degenerate nucleic acids, encoding the disclosed variants
and derivatives of the amino acid sequences. Thus, while each
particular nucleic acid sequence may not be written out herein, it
is understood that each and every sequence is in fact disclosed and
described herein through the disclosed amino acid sequences.
[0101] Also disclosed are bifunctional peptides, which contain the
homing peptide fused to a second peptide having a separate
function. Such bifunctional peptides have at least two functions
conferred by different portions of the full-length molecule and
can, for example, display anti-angiogenic activity or pro-apoptotic
activity in addition to the ability to home to a target.
[0102] Also disclosed are isolated multivalent peptides that
include at least two subsequences each independently containing a
peptide or amino acid segment. The multivalent peptide can have,
for example, at least three, at least five or at least ten of such
subsequences each independently containing a peptide. In particular
embodiments, the multivalent peptide can have two, three, four,
five, six, seven, eight, nine, ten, fifteen or twenty identical or
non-identical subsequences. This is in addition to the multiple
homing molecules and, for example, multiple membrane disrupting
molecules that can comprise the disclosed compositions. In a
further embodiment, the multivalent peptide can contain identical
subsequences, such as repeats of a specified amino acid sequence.
In a further embodiment, the multivalent peptide contains
contiguous identical or non-identical subsequences, which are not
separated by any intervening amino acids.
[0103] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids and have an activity such as selective
interaction with a target of the peptide upon which the
peptidomimetic is derived (see, for example, Goodman and Ro,
Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry
and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons
1995), pages 803-861).
[0104] A variety of peptidomimetics are known in the art including,
for example, peptide-like molecules which contain a constrained
amino acid, a non-peptide component that mimics peptide secondary
structure, or an amide bond isostere. A peptidomimetic that
contains a constrained, non-naturally occurring amino acid can
include, for example, an .alpha.-methylated amino acid;
.alpha.,.alpha..-dialkylglycine or .alpha.-aminocycloalkane
carboxylic acid; an N.sup..alpha.--C.sup..alpha. cyclized amino
acid; an N.sup..alpha..-methylated amino acid; a .beta.- or
.gamma.-amino cycloalkane carboxylic acid; an
.alpha.,.beta.-unsaturated amino acid; a .beta.,.beta.-dimethyl or
.beta.-methyl amino acid; a .beta.-substituted-2,3-methano amino
acid; an N--C.sup..epsilon. or C.sup..alpha.--C.sup..DELTA.
cyclized amino acid; a substituted proline or another amino acid
mimetic. A peptidomimetic which mimics peptide secondary structure
can contain, for example, a non-peptidic .alpha.-turn mimic;
.gamma.-turn mimic; mimic of .beta.-sheet structure; or mimic of
helical structure, each of which is well known in the art. A
peptidomimetic also can be a peptide-like molecule which contains,
for example, an amide bond isostere such as a retro-inverso
modification; reduced amide bond; methylenethioether or
methylene-sulfoxide bond; methylene ether bond; ethylene bond;
thioamide bond; trans-olefin or fluoroolefin bond;
1,5-disubstituted tetrazole ring; ketomethylene or
fluoroketomethylene bond or another amide isostere. One skilled in
the art understands that these and other peptidomimetics are
encompassed within the meaning of the term "peptidomimetic" as used
herein.
[0105] Methods for identifying a peptidomimetic are well known in
the art and include, for example, the screening of databases that
contain libraries of potential peptidomimetics. As an example, the
Cambridge Structural Database contains a collection of greater than
300,000 compounds that have known crystal structures (Allen et al.,
Acta Crystalloqr. Section B, 35:2331 (1979)). This structural
depository is continually updated as new crystal structures are
determined and can be screened for compounds having suitable
shapes, for example, the same shape as a disclosed peptide, as well
as potential geometrical and chemical complementarity to a target
molecule. Where no crystal structure of a peptide or a target
molecule that binds the peptide is available, a structure can be
generated using, for example, the program CONCORD (Rusinko et al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the
Available Chemicals Directory (Molecular Design Limited,
Information Systems; San Leandro Calif.), contains about 100,000
compounds that are commercially available and also can be searched
to identify potential peptidomimetics of a peptide, for example,
with activity in selectively interacting with cancerous cells.
6. Pharmaceutical Compositions and Carriers
[0106] The disclosed compositions can be administered in vivo
either alone or in a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material can be
administered to a subject, along with the composition disclosed
herein, without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art. The materials can be in solution, suspension (for
example, incorporated into microparticles, liposomes, or
cells).
i. Pharmaceutically Acceptable Carriers
[0107] The compositions disclosed herein can be used
therapeutically in combination with a pharmaceutically acceptable
carrier.
[0108] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,
an appropriate amount of a pharmaceutically-acceptable salt is used
in the formulation to render the formulation isotonic. Examples of
the pharmaceutically-acceptable carrier include, but are not
limited to, saline, Ringer's solution and dextrose solution. The pH
of the solution is preferably from about 5 to about 8, and more
preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers can be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0109] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0110] Pharmaceutical compositions can include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions can also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0111] The pharmaceutical composition can be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration can be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The disclosed antibodies can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally.
[0112] Parenteral formulations can include the active ingredient
combined with a pharmaceutically acceptable carrier, such as
sterile water or sterile isotonic saline. Such formulations may be
prepared, packaged, or sold in a form suitable for bolus
administration or for continuous administration. Injectable
formulations may be prepared, packaged, or sold in unit dosage
form, such as in ampules or in multi-dose containers containing a
preservative. Parenteral administration formulations include
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, reconsitutable dry (i.e. powder or granular) formulations,
and implantable sustained-release or biodegradable formulations.
Such formulations may also include one or more additional
ingredients including suspending, stabilizing, or dispersing
agents. Parenteral formulations may be prepared, packaged, or sold
in the form of a sterile injectable aqueous or oily suspension or
solution. Parenteral formulations may also include dispersing
agents, wetting agents, or suspending agents described herein.
Methods for preparing these types of formulations are known.
Sterile injectable formulations may be prepared using non-toxic
parenterally-acceptable diluents or solvents, such as water,
1,3-butane diol, Ringer's solution, isotonic sodium chloride
solution, and fixed oils such as synthetic monoglycerides or
diglycerides. Other parentally-administrable formulations include
microcrystalline forms, liposomal preparations, and biodegradable
polymer systems. Compositions for sustained release or implantation
may include pharmaceutically acceptable polymeric or hydrophobic
materials such as emulsions, ion exchange resins, sparingly soluble
polymers, and sparingly soluble salts.
[0113] Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0114] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0115] Compositions may be prepared, packaged, or sold in a buccal
formulation. Such formulations may be in the form of tablets,
powders, aerosols, atomized solutions, suspensions, or lozenges
made using known methods, and may contain from about 0.1% to about
20% (w/w) active ingredient with the balance of the formulation
containing an orally dissolvable or degradable composition and/or
one or more additional ingredients as described herein. Preferably,
powdered or aerosolized formulations have an average particle or
droplet size ranging from about 0.1 nanometers to about 200
nanometers when dispersed.
[0116] As used herein, "additional ingredients" include one or more
of the following: excipients, surface active agents, dispersing
agents, inert diluents, granulating agents, disintegrating agents,
binding agents, lubricating agents, sweetening agents, flavoring
agents, coloring agents, preservatives, physiologically degradable
compositions (e.g., gelatin), aqueous vehicles, aqueous solvents,
oily vehicles and oily solvents, suspending agents, dispersing
agents, wetting agents, emulsifying agents, demulcents, buffers,
salts, thickening agents, fillers, emulsifying agents,
antioxidants, antibiotics, antifungal agents, stabilizing agents,
and pharmaceutically acceptable polymeric or hydrophobic materials.
Other "additional ingredients" which may be included in the
pharmaceutical compositions are known. Suitable additional
ingredients are described in Remington's Pharmaceutical Sciences,
Mack Publishing Co., Genaro, ed., Easton, Pa. (1985).
[0117] Some of the compositions can be administered as a
pharmaceutically acceptable acid- or base-addition salt, formed by
reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
7. Delivery Systems
[0118] Expression vectors can comprise the DNA or RNA molecule of
the invention, wherein said expression vector is capable of
expressing a recombinant outer membrane protein of the invention
when present in a compatible host cell, and a host cell comprising
this expression vector.
[0119] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., 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 and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989)).
[0120] Delivery can be applied, in general, via local or systemic
routes of administration. Local administration includes virus
injection directly into the region or organ of interest, versus
intravenous (IV) or intraperitoneal (IP) injections (systemic)
aiming at viral delivery to multiple sites and organs via the blood
circulation. Previous research on the effects of local
administration demonstrated gene expression limited to the
site/organ of the injection, which did not extend to the rest of
the body (Daly et al., 1999a; Kordower et al., 1999). Furthermore,
previous studies have demonstrated successful global gene transfer
to multiple tissues and organs in rodents and primates following
viral IV and IP injections (Daly et al., 1999b; Tarntal et al.,
2001; McCormack et al., 2001; Lipschutz et al., 2001). Disclosed
herein IP injection of FIV(lacZ) in mice of adult (3 months old) as
well as of perinatal age (P4) resulted in global transfer and
expression of the reporter gene lacZ in brain, liver, spleen and
kidney. Also disclosed, the levels of expression achieved via IP
injections were superior to those acquired following local
administration directly into the liver.
[0121] As stated above, there are a number of compositions and
methods which can be used to deliver nucleic acids to cells, either
in vitro or in vivo. These methods and compositions can largely be
broken down into two classes: viral based delivery systems and
non-viral based delivery systems. For example, the nucleic acids
can be delivered through a number of direct delivery systems such
as, electroporation, lipofection, calcium phosphate precipitation,
plasmids, viral vectors, viral nucleic acids, phage nucleic acids,
phages, cosmids, or via transfer of genetic material in cells or
carriers such as cationic liposomes. Appropriate means for
transfection, including viral vectors, chemical transfectants, or
physico-mechanical methods such as electroporation and direct
diffusion of DNA, are described by, for example, Wolff, J. A., et
al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352,
815-818, (1991). Such methods are well known in the art and readily
adaptable for use with the compositions and methods described
herein. In certain cases, the methods will be modified to
specifically function with large DNA molecules. Further, these
methods can be used to target certain diseases and cell populations
by using the targeting characteristics of the carrier.
[0122] a) Nucleic Acid Based Delivery Systems
[0123] Transfer vectors can be any nucleotide construction used to
deliver genes into cells (e.g., a plasmid), or as part of a general
strategy to deliver genes, e.g., as part of recombinant retrovirus
or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
[0124] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids, such as P6 construct into
the cell without degradation and include a promoter yielding
expression of P6 encoding sequences in the cells into which it is
delivered. In some embodiments the vectors for the P6 constructs
are derived from either a virus, retrovirus, or lentivirus. Viral
vectors can be, for example, Adenovirus, Adeno-associated virus,
Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal
trophic virus, Sindbis and other RNA viruses, including these
viruses with the HIV backbone, and lentiviruses. Also preferred are
any viral families which share the properties of these viruses
which make them suitable for use as vectors. Retroviruses include
Murine Maloney Leukemia virus, MMLV, and retroviruses that express
the desirable properties of MMLV as a vector. Retroviral vectors
are able to carry a larger genetic payload, i.e., a transgene, such
as, the disclosed P6 constructs or marker gene, than other viral
vectors, and for this reason are commonly used vectors. However,
they are not as useful in non-proliferating cells. Adenovirus
vectors are relatively stable and easy to work with, have high
titers, and can be delivered in aerosol formulation, and can
transfect non-dividing cells. Pox viral vectors are large and have
several sites for inserting genes, they are thermostable and can be
stored at room temperature. A preferred embodiment is a viral
vector, which has been engineered so as to suppress the immune
response of the host organism, elicited by the viral antigens.
Preferred vectors of this type will carry coding regions for
Interleukin 8 or 10.
[0125] Viral vectors can have higher transaction (ability to
introduce genes) abilities than chemical or physical methods to
introduce genes into cells. Typically, viral vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase
III transcript, inverted terminal repeats necessary for replication
and encapsidation, and promoters to control the transcription and
replication of the viral genome. When engineered as vectors,
viruses typically have one or more of the early genes removed and a
gene or gene/promotor cassette is inserted into the viral genome in
place of the removed viral DNA. Constructs of this type can carry
up to about 8 kb of foreign genetic material. The necessary
functions of the removed early genes are typically supplied by cell
lines which have been engineered to express the gene products of
the early genes in trans.
a. Retroviral Vectors
[0126] A retrovirus is an animal virus belonging to the virus
family of Retroviridae, including any types, subfamilies, genus, or
tropisms. Retroviral vectors, in general, are described by Verma,
I. M., Retroviral vectors for gene transfer. In Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985),
which is incorporated by reference herein. Examples of methods for
using retroviral vectors for gene therapy are described in U.S.
Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and
WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the
teachings of which are incorporated herein by reference.
[0127] A retrovirus is essentially a package which has packed into
it nucleic acid cargo. The nucleic acid cargo carries with it a
packaging signal, which ensures that the replicated daughter
molecules will be efficiently packaged within the package coat. In
addition to the package signal, there are a number of molecules
which are needed in cis, for the replication, and packaging of the
replicated virus. Typically a retroviral genome, contains the gag,
pol, and env genes which are involved in the making of the protein
coat. It is the gag, pol, and env genes which are typically
replaced by the foreign DNA that it is to be transferred to the
target cell. Retrovirus vectors typically contain a packaging
signal for incorporation into the package coat, a sequence which
signals the start of the gag transcription unit, elements necessary
for reverse transcription, including a primer binding site to bind
the tRNA primer of reverse transcription, terminal repeat sequences
that guide the switch of RNA strands during DNA synthesis, a purine
rich sequence 5' to the 3' LTR that serve as the priming site for
the synthesis of the second strand of DNA synthesis, and specific
sequences near the ends of the LTRs that enable the insertion of
the DNA state of the retrovirus to insert into the host genome. The
removal of the gag, pol, and env genes allows for about 8 kb of
foreign sequence to be inserted into the viral genome, become
reverse transcribed, and upon replication be packaged into a new
retroviral particle. This amount of nucleic acid is sufficient for
the delivery of a one to many genes depending on the size of each
transcript. It is preferable to include either positive or negative
selectable markers along with other genes in the insert.
[0128] Since the replication machinery and packaging proteins in
most retroviral vectors have been removed (gag, pol, and env), the
vectors are typically generated by placing them into a packaging
cell line. A packaging cell line is a cell line which has been
transfected or transformed with a retrovirus that contains the
replication and packaging machinery, but lacks any packaging
signal. When the vector carrying the DNA of choice is transfected
into these cell lines, the vector containing the gene of interest
is replicated and packaged into new retroviral particles, by the
machinery provided in cis by the helper cell. The genomes for the
machinery are not packaged because they lack the necessary
signals.
b. Adenoviral Vectors
[0129] The construction of replication-defective adenoviruses has
been described (Berkner et al., J. Virology 61:1213-1220 (1987);
Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et
al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology
61:1226-1239 (1987); Zhang "Generation and identification of
recombinant adenovirus by liposome-mediated transfection and PCR
analysis" BioTechniques 15:868-872 (1993)). The benefit of the use
of these viruses as vectors is that they are limited in the extent
to which they can spread to other cell types, since they can
replicate within an initial infected cell, but are unable to form
new infectious viral particles. Recombinant adenoviruses have been
shown to achieve high efficiency gene transfer after direct, in
vivo delivery to airway epithelium, hepatocytes, vascular
endothelium, CNS parenchyma and a number of other tissue sites
(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.
Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092
(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle,
Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem.
267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993);
Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation
Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10
(1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J.
Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology
74:501-507 (1993)). Recombinant adenoviruses achieve gene
transduction by binding to specific cell surface receptors, after
which the virus is internalized by receptor-mediated endocytosis,
in the same manner as wild type or replication-defective adenovirus
(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and
Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J.
Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655
(1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et
al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell
73:309-319 (1993)).
[0130] A viral vector can be one based on an adenovirus which has
had the E1 gene removed and these virions are generated in a cell
line such as the human 293 cell line. In another preferred
embodiment both the E1 and E3 genes are removed from the adenovirus
genome.
c. Adeno-Associated Viral Vectors
[0131] Another type of viral vector is based on an adeno-associated
virus (AAV). This defective parvovirus is a preferred vector
because it can infect many cell types and is nonpathogenic to
humans. AAV type vectors can transport about 4 to 5 kb and wild
type AAV is known to stably insert into chromosome 19. Vectors
which contain this site specific integration property are
preferred. An especially preferred embodiment of this type of
vector is the P4.1 C vector produced by Avigen, San Francisco,
Calif., which can contain the herpes simplex virus thymidine kinase
gene, HSV-tk, and/or a marker gene, such as the gene encoding the
green fluorescent protein, GFP.
[0132] In another type of AAV virus, the AAV contains a pair of
inverted terminal repeats (ITRs) which flank at least one cassette
containing a promoter which directs cell-specific expression
operably linked to a heterologous gene. Heterologous in this
context refers to any nucleotide sequence or gene which is not
native to the AAV or B19 parvovirus.
[0133] Typically the AAV and B19 coding regions have been deleted,
resulting in a safe, noncytotoxic vector. The AAV ITRs, or
modifications thereof, confer infectivity and site-specific
integration, but not cytotoxicity, and the promoter directs
cell-specific expression. U.S. Pat. No. 6,261,834 is herein
incorporated by reference for material related to the AAV
vector.
[0134] The vectors of the present invention thus provide DNA
molecules which are capable of integration into a mammalian
chromosome without substantial toxicity.
[0135] The inserted genes in viral and retroviral usually contain
promoters, and/or enhancers to help control the expression of the
desired gene product. A promoter is generally a sequence or
sequences of DNA that function when in a relatively fixed location
in regard to the transcription start site. A promoter contains core
elements required for basic interaction of RNA polymerase and
transcription factors, and can contain upstream elements and
response elements.
d. Lentiviral Vectors
[0136] The vectors can be lentiviral vectors, including but not
limited to, SW vectors, HIV vectors or a hybrid construct of these
vectors, including viruses with the HIV backbone.
[0137] These vectors also include first, second and third
generation lentiviruses. Third generation lentiviruses have
lentiviral packaging genes split into at least 3 independent
plasmids or constructs. Also vectors can be any viral family that
shares the properties of these viruses which make them suitable for
use as vectors. Lentiviral vectors are a special type of retroviral
vector which are typically characterized by having a long
incubation period for infection. Furthermore, lentiviral vectors
can infect non-dividing cells. Lentiviral vectors are based on the
nucleic acid backbone of a virus from the lentiviral family of
viruses. Typically, a lentiviral vector contains the 5' and 3' LTR
regions of a lentivirus, such as SIV and HIV. Lentiviral vectors
also typically contain the Rev Responsive Element (RRE) of a
lentivirus, such as SW and HIV.
(A) Feline Immunodeficiency Viral Vectors
[0138] One type of vector that the disclosed constructs can be
delivered in is the VSV-G pseudotyped Feline Immunodeficiency Virus
system developed by Poeschla et al. (1998). This lentivirus has
been shown to efficiently infect dividing, growth arrested as well
as post-mitotic cells. Furthermore, due to its lentiviral
properties, it allows for incorporation of the transgene into the
host's genome, leading to stable gene expression. This is a
3-vector system, whereby each confers distinct instructions: the
FIV vector carries the transgene of interest and lentiviral
apparatus with mutated packaging and envelope genes. A vesicular
stomatitis virus G-glycoprotein vector (VSV-G; Burns et al., 1993)
contributes to the formation of the viral envelope in trans. The
third vector confers packaging instructions in trans (Poeschla et
al., 1998). FIV production is accomplished in vitro following
co-transfection of the aforementioned vectors into 293-T cells. The
FIV-rich supernatant is then collected, filtered and can be used
directly or following concentration by centrifugation. Titers
routinely range between 104-107 bfu/ml.
e. Packaging Vectors
[0139] As discussed above, retroviral vectors are based on
retroviruses which contain a number of different sequence elements
that control things as diverse as integration of the virus,
replication of the integrated virus, replication of un-integrated
virus, cellular invasion, and packaging of the virus into
infectious particles. While the vectors in theory could contain all
of their necessary elements, as well as an exogenous gene element
(if the exogenous gene element is small enough) typically many of
the necessary elements are removed. Since all of the packaging and
replication components have been removed from the typical
retroviral, including lentiviral, vectors which will be used within
a subject, the vectors need to be packaged into the initial
infectious particle through the use of packaging vectors and
packaging cell lines. Typically retroviral vectors have been
engineered so that the myriad functions of the retrovirus are
separated onto at least two vectors, a packaging vector and a
delivery vector. This type of system then requires the presence of
all of the vectors providing all of the elements in the same cell
before an infectious particle can be produced. The packaging vector
typically carries the structural and replication genes derived from
the retrovirus, and the delivery vector is the vector that carries
the exogenous gene element that is preferably expressed in the
target cell. These types of systems can split the packaging
functions of the packaging vector into multiple vectors, e.g.,
third-generation lentivirus systems. Dull, T. et al., "A
Third-generation lentivirus vector with a conditional packaging
system" J. Virol 72(11):8463-71 (1998)
[0140] Retroviruses typically contain an envelope protein (env).
The Env protein is in essence the protein which surrounds the
nucleic acid cargo. Furthermore cellular infection specificity is
based on the particular Env protein associated with a typical
retrovirus. In typical packaging vector/delivery vector systems,
the Env protein is expressed from a separate vector than for
example the protease (pro) or integrase (in) proteins.
(A) Packaging Cell Lines
[0141] The vectors are typically generated by placing them into a
packaging cell line. A packaging cell line is a cell line which has
been transfected or transformed with a retrovirus that contains the
replication and packaging machinery, but lacks any packaging
signal. When the vector carrying the DNA of choice is transfected
into these cell lines, the vector containing the gene of interest
is replicated and packaged into new retroviral particles, by the
machinery provided in cis by the helper cell. The genomes for the
machinery are not packaged because they lack the necessary signals.
One type of packaging cell line is a 293 cell line.
f. Large Payload Viral Vectors
[0142] Molecular genetic experiments with large human herpesviruses
have provided a means whereby large heterologous DNA fragments can
be cloned, propagated and established in cells permissive for
infection with herpesviruses (Sun et al., Nature genetics 8: 33-41,
1994; Cotter and Robertson. Curr Opin Mol Ther 5: 633-644, 1999).
These large DNA viruses (herpes simplex virus (HSV) and
Epstein-Barr virus (EBV), have the potential to deliver fragments
of human heterologous DNA>150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected B
cells as episomal DNA. Individual clones carried human genomic
inserts up to 330 kb appeared genetically stable. The maintenance
of these episomes requires a specific EBV nuclear protein, EBNA1,
constitutively expressed during infection with EBV. Additionally,
these vectors can be used for transfection, where large amounts of
protein can be generated transiently in vitro. Herpesvirus amplicon
systems are also being used to package pieces of DNA>220 kb and
to infect cells that can stably maintain DNA as episomes.
[0143] Other useful systems include, for example, replicating and
host-restricted non-replicating vaccinia virus vectors.
ii. Non-Nucleic Acid Based Systems
[0144] The disclosed compositions can be delivered to the target
cells in a variety of ways. For example, the compositions can be
delivered through electroporation, or through lipofection, or
through calcium phosphate precipitation. The delivery mechanism
chosen will depend in part on the type of cell targeted and whether
the delivery is occurring for example in vivo or in vitro.
[0145] Thus, the compositions can comprise, in addition to the
disclosed constructs or vectors for example, lipids such as
liposomes, such as cationic liposomes (e.g., DOTMA, DOPE,
DC-cholesterol) or anionic liposomes. Liposomes can further
comprise proteins to facilitate targeting a particular cell, if
desired. Administration of a composition comprising a compound and
a cationic liposome can be administered to the blood afferent to a
target organ or inhaled into the respiratory tract to target cells
of the respiratory tract. Regarding liposomes, see, e.g., Brigham
et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et
al. Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987); U.S. Pat. No.
4,897,355. Furthermore, the compound can be administered as a
component of a microcapsule that can be targeted to specific cell
types, such as macrophages, or where the diffusion of the compound
or delivery of the compound from the microcapsule is designed for a
specific rate or dosage.
[0146] In the methods described above which include the
administration and uptake of exogenous DNA into the cells of a
subject (i.e., gene transduction or transfection), delivery of the
compositions to cells can be via a variety of mechanisms. As one
example, delivery can be via a liposome, using commercially
available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE
(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc.
Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
Wis.), as well as other liposomes developed according to procedures
standard in the art. In addition, the nucleic acid or vector of
this invention can be delivered in vivo by electroporation, the
technology for which is available from Genetronics, Inc. (San
Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx
Pharmaceutical Corp., Tucson, Ariz.).
[0147] The materials can be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These can
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). These techniques can be used for a variety
of other specific cell types. Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)). Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral
integration systems can also be incorporated into nucleic acids
which are to be delivered using a non-nucleic acid based system of
deliver, such as a liposome, so that the nucleic acid contained in
the delivery system can be come integrated into the host
genome.
[0148] Other general techniques for integration into the host
genome include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target
nucleic acid takes place, causing the delivered nucleic acid to be
integrated into the host genome. These systems and the methods
necessary to promote homologous recombination are known to those of
skill in the art.
8. Expression Systems
[0149] The nucleic acids that are delivered to cells typically
contain expression controlling systems. For example, the inserted
genes in viral and retroviral systems usually contain promoters,
and/or enhancers to help control the expression of the desired gene
product. A promoter is generally a sequence or sequences of DNA
that function when in a relatively fixed location in regard to the
transcription start site. A promoter contains core elements
required for basic interaction of RNA polymerase and transcription
factors, and can contain upstream elements and response
elements.
i. Promoters and Enhancers
[0150] Preferred promoters controlling transcription from vectors
in mammalian host cells can be obtained from various sources, for
example, the genomes of viruses such as: polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. beta actin promoter. The early and late promoters
of the SV40 virus are conveniently obtained as an SV40 restriction
fragment which also contains the SV40 viral origin of replication
(Fiers et al., Nature, 273: 113 (1978)). The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:
355-360 (1982)). Of course, promoters from the host cell or related
species also are useful herein.
[0151] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.
78: 993 (1981)) or 3' (Lusky, M. L., et al., Mol. Cell. Bio. 3:
1108 (1983)) to the transcription unit. Furthermore, enhancers can
be within an intron (Banerji, J. L. et al., Cell 33: 729 (1983)) as
well as within the coding sequence itself (Osborne, T. F., et al.,
Mol. Cell. Bio. 4: 1293 (1984)). They are usually between 10 and
300 by in length, and they function in cis. Enhancers function to
increase transcription from nearby promoters. Enhancers also often
contain response elements that mediate the regulation of
transcription. Promoters can also contain response elements that
mediate the regulation of transcription. Enhancers often determine
the regulation of expression of a gene. While many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, alpha-fetoprotein and insulin), typically one will use an
enhancer from a eukaryotic cell virus for general expression.
Preferred examples are the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0152] The promoter and/or enhancer can be specifically activated
either by light or specific chemical events which trigger their
function. Systems can be regulated by reagents such as tetracycline
and dexamethasone. There are also ways to enhance viral vector gene
expression by exposure to irradiation, such as gamma irradiation,
or alkylating chemotherapy drugs.
[0153] In certain embodiments the promoter and/or enhancer region
can act as a constitutive promoter and/or enhancer to maximize
expression of the region of the transcription unit to be
transcribed. In certain constructs the promoter and/or enhancer
region be active in all eukaryotic cell types, even if it is only
expressed in a particular type of cell at a particular time. A
preferred promoter of this type is the CMV promoter (650 bases).
Other preferred promoters are SV40 promoters, cytomegalovirus (full
length promoter), and retroviral vector LTF.
[0154] It has been shown that all specific regulatory elements can
be cloned and used to construct expression vectors that are
selectively expressed in specific cell types such as melanoma
cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to selectively express genes in cells of glial origin.
[0155] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) can also
contain sequences necessary for the termination of transcription
which can affect mRNA expression. These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding tissue factor protein. The 3' untranslated regions also
include transcription termination sites. It is preferred that the
transcription unit also contains a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that homologous
polyadenylation signals be used in the transgene constructs. In
certain transcription units, the polyadenylation region is derived
from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain
other standard sequences alone or in combination with the above
sequences improve expression from, or stability of, the
construct.
a. Constitutive Promoters
[0156] In certain embodiments the promoters are constitutive
promoters. This can be any promoter that causes transcription
regulation in the absence of the addition of other factors.
Examples of this type of promoter are the CMV promoter and the beta
actin promoter, as well as others discussed herein. In certain
embodiments the promoter can consist of fusions of one or more
different types of promoters. For example, the regulatory regions
of the CMV promoter and the beta actin promoter are well known and
understood, examples, of which are disclosed herein. Parts of these
promoters can be fused together to, for example, produce a CMV-beta
actin fusion promoter. It is understood that this type of promoter
has a CMV component and a beta actin component. These components
can function independently as promoters, and thus, are themselves
considered beta actin promoters and CMV promoters. A promoter can
be any portion of a known promoter that causes promoter activity.
It is well understood that many promoters, including the CMV and
Beta Actin promoters have functional domains which are understood
and that these can be used as a beta actin promoter or CMV
promoter. Furthermore, these domains can be determined. There are
many CMV promoter variations that exist, as well as beta actin
promoters, and fusion promoters. These promoters can be compared,
and for example, functional regions delineated, as described
herein. Furthermore, each of these sequences can function
independently or together in any combination to provide a promoter
region for the disclosed nucleic acids.
b. Non-Constitutive Promoters
[0157] The promoters can also be non-constitutive promoters, such
as cell specific promoters. These are promoters that are turned on
at specific time in development or stage or a particular type of
cell, such as a cardiac cell, or neural cell, or a bone cell. Some
examples of cell specific promoters are, the neural enolase
specific promoter, (NSE) the COLL1A1 procollagen promoter, and the
CD11b promoter (PBMC-microglia/macrophage/monocyte specific
promoter.
[0158] It is understood that the recombinant systems can be
expressed in a tissue-specific manner. It is understood that tissue
specific expression can occur due to the presence of a
tissue-specific promoter. Typically, proteins under control of a
tissue-specific promoter are transcribed when the promoter becomes
active by virtue of being present in the tissue for which it is
specific. Therefore, all cells can encode for a particular gene
without global expression. As such, labeled proteins can be shown
to be present in certain tissues without expression in other nearby
tissues that may complicate results or expression of proteins in
tissues where expression may be detrimental to the host. Disclosed
are methods wherein the cre recombinase is under the control of the
EIIA promoter, a promoter specific for breast tissue, such as the
WAP promoter, a promoter specific for ovarian tissue, such as the
ACTB promoter, or a promoter specific for bone tissue, such as
osteocalcin. Any tissues specific promoter can be used. Promoters
specific for prostate, testis, and neural are also disclosed.
Examples of some tissue-specific promoters include but are not
limited to MUC1, EIIA, ACTB, WAP, bHLH-EC2, HOXA-1,
Alpha-fetoprotein (AFP), opsin, CR1/2, Fc-.quadrature.-Receptor 1
(Fc-.quadrature.-R1), MMTVD-LTR, the human insulin promoter,
Pdha-2, rat neuron-specific enolase. For example, use of the AFP
promoter creates specificity for the liver. Another example, HOXA-1
is a neuronal tissue specific promoter, and as such, proteins
expressed under the control of HOXA-1 are only expressed in
neuronal tissue. (All of which are herein incorporated by reference
at least for the sequence of the promoters and related
sequences.)
[0159] Other cell specific promoters can be found in (Sutcliffe, J.
G. (1988), Ann. Rev. Neuroscience 11, 157-198). For example, when
transfecting nerve cells, there are a variety of nerve specific
promoters, such as the neuron specific enolase promoter. Other
examples of neuron specific promoters would be the Tau promoter,
Synapsin I (Hoesche, C., Sauerwald, A., et al., (1993) J. Biol.
Chem. 268, 26494-26502. and II (Chin, L.-S et al., (1994), J. Biol.
Chem. 269, 18507-18513) promoters, the amino acid decarboxylase
(AADC) (Albert, V., et al., (1992), Proc. Natl. Acad. Sci. 89,
12053-12057) and FE65 (Faraonio, R., et al., (1994), Nucl. Acids
Res. 22, 4876-4883) promoters. Other nerve specific promoters
include, the promoter for the WT1 gene (Fraizer, G, et al., (1994),
J. Biol. Chem. 269, 8892-8900), nuerofilament light chain promoter
(Yazdanbakhsh, K., et al., (1993) Nucl. Acids Res. 21, 455-461),
and the glial fibrillary acidic protein, (Kaneko, R. & Sueoka,
N. (1993) Proc. Natl. Acad. Sci. 90, 4698-4702). (All of which are
herein incorporated by reference at least for the sequence of the
promoters and related sequences.)
[0160] Expression of the transgene can be targeted selectively to
neurons by cloning a neuron specific promoter, such as the NSE
promoter as disclosed herein (Liu H. et al., Journal of
Neuroscience. 23(18):7143-54, 2003); tyrosine hydroxylase promoter
(Kessler M A. et al., Brain Research. Molecular Brain Research.
112(1-2):8-23, 2003); myelin basic protein promoter (Kessler M A.
et al Biochemical & Biophysical Research Communications.
288(4):809-18, 2001); glial fibrillary acidic protein promoter
(Nolte C. et al., GLIA. 33(1):72-86, 2001); neurofilaments gene
(heavy, medium, light) promoters (Yaworsky P J. et al., Journal of
Biological Chemistry. 272(40):25112-20, 1997) (All of which are
herein incorporated by reference at least for the sequence of the
promoters and related sequences.) The NSE promoter is disclosed in
Peel A L. et al., Gene Therapy. 4(1):16-24, 1997) (SEQ ID NO:69)
(pTR-NT3myc; Powell Gene Therapy Center, University of Florida,
Gainesville Fla.).
ii. Markers
[0161] The viral vectors can include nucleic acid sequence encoding
a marker product. This marker product is used to determine if the
gene has been delivered to the cell and once delivered is being
expressed. Preferred marker genes are the E. Coli lacZ gene, which
encodes .beta.-galactosidase, and green fluorescent protein.
[0162] In some embodiments the marker can be a selectable marker.
Examples of suitable selectable markers for mammalian cells are
dihydrofolate reductase (DHFR), thymidine kinase, neomycin,
neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian
host cell, the transformed mammalian host cell can survive if
placed under selective pressure. There are two widely used distinct
categories of selective regimes. The first category is based on a
cell's metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine
or hypoxanthine. Because these cells lack certain genes necessary
for a complete nucleotide synthesis pathway, they cannot survive
unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes,
thus altering their growth requirements. Individual cells which
were not transformed with the DHFR or TK gene will not be capable
of survival in non-supplemented media.
[0163] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327
(1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science
209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mal. Cell.
Biol. 5: 410-413 (1985)). The three examples employ bacterial genes
under eukaryotic control to convey resistance to the appropriate
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin, respectively. Others include the neomycin analog G418
and puramycin.
iii. Post Transcriptional Regulatory Elements
[0164] The disclosed vectors can also contain post-transcriptional
regulatory elements.
[0165] Post-transcriptional regulatory elements can enhance mRNA
stability or enhance translation of the transcribed mRNA. An
exemplary post-transcriptional regulatory sequence is the WPRE
sequence isolated from the woodchuck hepatitis virus. (Zufferey R,
et al., "Woodchuck hepatitis virus post-transcriptional regulatory
element enhances expression of transgenes delivered by retroviral
vectors," J Virol; 73:2886-92 (1999)). Post-transcriptional
regulatory elements can be positioned both 3' and 5' to the
exogenous gene, but it is preferred that they are positioned 3' to
the exogenous gene.
iv. Transduction Efficiency Elements
[0166] Transduction efficiency elements are sequences that enhance
the packaging and transduction of the vector. These elements
typically contain polypurine sequences. An example of a
transduction efficiency element is the ppt-cts sequence that
contains the central polypurine tract (ppt) and central terminal
site (cts) from the FIV. These sequences are in the disclosed FIV
sequences herein. Each retrovirus and lentivirus can have there own
ppt-cts.
v. 3' Untranslated Regions
[0167] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) can also
contain sequences necessary for the termination of transcription
which can affect mRNA expression. These 3' untranslated regions are
transcribed as polyadenylated segments in the untranslated portion
of the mRNA encoding the exogenous gene. The 3' untranslated
regions also include transcription termination sites. The
transcription unit also can contain a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. Homologous polyadenylation signals
can be used in the transgene constructs. In an embodiment of the
transcription unit, the polyadenylation region is derived from the
SV40 early polyadenylation signal and consists of about 400 bases.
Transcribed units can contain other standard sequences alone or in
combination with the above sequences improve expression from, or
stability of, the construct.
9. Sequence Similarities
[0168] It is understood that as discussed herein the use of the
terms homology and identity mean the same thing as similarity.
Thus, for example, if the use of the word homology is used between
two non-natural sequences it is understood that this is not
necessarily indicating an evolutionary relationship between these
two sequences, but rather is looking at the similarity or
relatedness between their nucleic acid sequences. Many of the
methods for determining homology between two evolutionarily related
molecules are routinely applied to any two or more nucleic acids or
proteins for the purpose of measuring sequence similarity
regardless of whether they are evolutionarily related or not.
[0169] In general, it is understood that one way to define any
known variants and derivatives or those that might arise, of the
disclosed genes and proteins herein, is through defining the
variants and derivatives in terms of homology to specific known
sequences. This identity of particular sequences disclosed herein
is also discussed elsewhere herein. In general, variants of genes
and proteins herein disclosed typically have at least, about 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology
to the stated sequence or the native sequence. Those of skill in
the art readily understand how to determine the homology of two
proteins or nucleic acids, such as genes. For example, the homology
can be calculated after aligning the two sequences so that the
homology is at its highest level.
[0170] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
can be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0171] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods can differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity, and be disclosed herein.
[0172] For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences
that have the recited homology as calculated by any one or more of
the calculation methods described above. For example, a first
sequence has 80 percent homology, as defined herein, to a second
sequence if the first sequence is calculated to have 80 percent
homology to the second sequence using the Zuker calculation method
even if the first sequence does not have 80 percent homology to the
second sequence as calculated by any of the other calculation
methods. As another example, a first sequence has 80 percent
homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second
sequence using both the Zuker calculation method and the Pearson
and Lipman calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by
the Smith and Waterman calculation method, the Needleman and Wunsch
calculation method, the Jaeger calculation methods, or any of the
other calculation methods. As yet another example, a first sequence
has 80 percent homology, as defined herein, to a second sequence if
the first sequence is calculated to have 80 percent homology to the
second sequence using each of calculation methods (although, in
practice, the different calculation methods will often result in
different calculated homology percentages).
10. Peptide Synthesis
[0173] The compositions disclosed herein and the compositions
necessary to perform the disclosed methods can be made using any
method known to those of skill in the art for that particular
reagent or compound unless otherwise specifically noted.
[0174] One method of producing the disclosed proteins is to link
two or more peptides or polypeptides together by protein chemistry
techniques. For example, peptides or polypeptides can be chemically
synthesized using currently available laboratory equipment using
either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to the disclosed
proteins, for example, can be synthesized by standard chemical
reactions. For example, a peptide or polypeptide can be synthesized
and not cleaved from its synthesis resin whereas the other fragment
of a peptide or protein can be synthesized and subsequently cleaved
from the resin, thereby exposing a terminal group which is
functionally blocked on the other fragment. By peptide condensation
reactions, these two fragments can be covalently joined via a
peptide bond at their carboxyl and amino termini, respectively, to
form an antibody, or fragment thereof. (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY (which is herein incorporated by reference
at least for material related to peptide synthesis). Alternatively,
the peptide or polypeptide is independently synthesized in vivo as
described herein. Once isolated, these independent peptides or
polypeptides can be linked to form a peptide or fragment thereof
via similar peptide condensation reactions.
[0175] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0176] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
11. Kits
[0177] Disclosed herein are kits that are drawn to reagents that
can be used in practicing the methods disclosed herein. The kits
can include any reagent or combination of reagent discussed herein
or that would be understood to be required or beneficial in the
practice of the disclosed methods. For example, the kits could
include the P6 vaccine.
Methods
[0178] A. Methods of Treating with Combination Therapies
[0179] The disclosed methods can comprise treating an individual
with one of the disclosed compositions, such as a P6 vaccine, in
combination with a therapeutic agent. The therapeutic agent can be
a therapeutic polypeptide, peptide (including one or more peptides
of the invention), a nucleic acid encoding a therapeutic
polypeptide, an anti-inflammatory agent, a biological and/or small
molecule targeting agent, an immunomodulatory agent, or a
combination thereof, for example. The therapeutic agent may be
administered simultaneously with the vaccine, or administered at a
different time than the vaccine.
[0180] Combination therapies can provide one or more therapeutic
benefits. The therapies can have a synergistic effect wherein the
presence of multiple therapies is more than the sum of each
individual therapy. There can be one therapeutic effect that is
enhanced by the presence of a second therapy.
[0181] The treatment of subjects can include the disclosed
compositions alone or in combination with a therapeutic agent. The
therapeutic agent can be any of the therapeutic agents disclosed
herein. The therapeutic agent can be but is not limited to an
anti-viral, anti-bacterial or anti-inflammatory agent.
[0182] The disclosed compositions can be administered as
multivalent subunit vaccines in combination with antigens from
other proteins of H. influenzae to achieve an enhanced bactericidal
activity. They can also be administered in combination with
polysaccharide antigens, for example the PRP capsular
polysaccharide (preferably conjugated to a protein such as tetanus
toxoid) of H. influenzae b. For combined administration with
epitopes of other proteins, the protein of the invention is either
administered separately, as a mixture (for instance within an outer
membrane vesicle preparation) or as a conjugate or genetic fusion
polypeptide. The conjugate is formed by standard techniques for
coupling proteinaceous materials. The proteins of the invention can
be used in conjunction with antigens of other organisms (e.g.
encapsulated or nonencapsulated, bacteria, viruses, fungi and
parasites). For example, the proteins of the invention are useful
in conjunction with antigens of other microorganisms implicated in
otitis media or other diseases. These include Streptococcus
pneumoniae, Streptococcus pyrogenes group A, Staphylococcus aureus,
respiratory syncytial virus and Moraxella catarrhalis
[0183] The compositions can be used in combination vaccines which
provide protection against a range of different pathogens. Many
pediatric vaccines are now given as a combination vaccine so as to
reduce the number of injections a child has to receive. Thus for
pediatric vaccines, other antigens from other pathogens may be
formulated with the compositions disclosed herein. For example the
disclosed compositions can be formulated with (or administered
separately but at the same time) the well known `trivalent`
combination vaccine comprising Diphtheria toxoid (DT), tetanus
toxoid (TT), and pertussis components [typically detoxified
Pertussis toxoid (PT) and filamentous haemagglutinin (FHA) with
optional pertactin (PRN) and/or agglutinin 1+2] for example, the
marketed vaccine INFANRIX-DTPa.TM. (SmithKlineBeecham Biologicals)
which contains DT, TT, PT, FHA and PRN antigens, or with a whole
cell pertussis component for example as marketed by
SmithKlineBeecham Biologicals s.a., as Tritanrix.TM.. The combined
vaccine may also comprise other antigens, such as Hepatitis B
surface antigen (HBsAg), Polio virus antigens (for instance
inactivated trivalent polio virus--IPV), Moraxella catarrhalis
outer membrane proteins, non-typeable Haemophilus influenzae
proteins, N. meningitidis B outer membrane proteins. Examples of
other non-typeable Haemophilus influenzae antigens which can be
included in a combination vaccine (especially for the prevention of
otitis media) include: Fimbrin protein [(U.S. Pat. No.
5,766,608-Ohio State Research Foundation)] and fusions comprising
peptides therefrom [eg LB1(f) peptide fusions; U.S. Pat. No.
5,843,464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)];
TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4;
Hap; D15 (WO 94/12641); P2; and P5 (WO 94/26304).
B. Methods of Administering
[0184] The compositions can be administered in a number of ways
depending on whether local or systemic treatment is desired, and on
the area to be treated. Administration can be topically (including
ophthalmically, vaginally, rectally, intranasally), orally, by
inhalation, or parenterally, for example by intravenous drip,
subcutaneous, intraperitoneal or intramuscular injection. The
disclosed compositions can be administered intravenously,
intraperitoneally, intramuscularly, subcutaneously, intracavity, or
transdermally.
[0185] The compositions provided herein may be administered in a
physiologically acceptable carrier to a host. Preferred methods of
administration include systemic or direct administration to a cell.
The compositions can be administered to a cell or subject, as is
generally known in the art for gene therapy applications. In gene
therapy applications, the compositions are introduced into cells in
order to transfect an organelle. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or RNA.
[0186] The modified complex compositions can be combined in
admixture with a pharmaceutically acceptable carrier vehicle.
Therapeutic formulations are prepared for storage by mixing the
active ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween, Pluronics or PEG.
1. Parental Administration
[0187] The compositions of the present disclosure can be
administered parenterally. As used herein, "parenteral
administration" is characterized by administering a pharmaceutical
composition through a physical breach of a subject's tissue.
Parenteral administration includes administering by injection,
through a surgical incision, or through a tissue-penetrating
non-surgical wound, and the like. In particular, parenteral
administration includes subcutaneous, intraperitoneal, intravenous,
intraarterial, intramuscular, intrasternal injection, and kidney
dialytic infusion techniques.
2. Dosages
[0188] Dosages and desired concentrations of the pharmaceutical
compositions of the present disclosure may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0189] The amount or dose of the material administered should be
sufficient to affect a therapeutic or prophylactic response in a
subject over a reasonable time frame. For example, the dose of the
material should be sufficient to treat a bacterial infection. The
dose should be sufficient to stimulate the immune response and/or
treat or prevent AOM in children prone to otitis media.
[0190] Many assays for determining an administered dose are known
in the art. For purposes of the present methods, an assay which
comprises comparing the bactericidal antibodies in response to
several different doses of a substance (i.e. P6 vaccine) to a set
of mammals can be performed. The dose also can be determined by the
existence, nature and extent of any adverse side effects that might
accompany the administration. A variety of factors, such as age,
body weight, general health, diet, sex, material to be
administered, route of administration, and the severity of the
condition being treated can be considered when determining
dosage.
3. Administration of Multiple Compositions
[0191] Simultaneous administration of two compositions, such as a
P6 vaccine and a therapeutic, means that the compositions are
administered at the same time. To be administered at the same time
means that both compositions are administered together.
Administering the compositions together involves formulating them
in a compatible carrier. Simultaneous administration can also refer
to administering one composition in one formulation and then
immediately administering the other composition. Simultaneous
administration is the administration of two or more compositions
within 30 minutes of each other.
[0192] The vaccine and the therapeutic can also be administered
consecutively. Consecutive administration refers to separate,
individual formulations for each composition. The compositions can
be administered in any order: the conjugate first or the second
therapeutic first. The term consecutive administration refers to
administration of one composition and then at least 30 minutes
later administering the other composition. The consecutive
administration can be at least 30 minutes, 45 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 18 hours, 1
day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14
days or 30 days from the administration of the first
composition.
DEFINITIONS
[0193] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
1. A, an the
[0194] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
2. AOM
[0195] AOM was diagnosed by pneumatic otoscopy by validated
otoscopists, when children with acute onset of otalgia had tympanic
membranes (TMs) that were: (1) bulging or full; and (2) a cloudy or
purulent effusion was observed, or the TM was completely opacified;
and (3) TM mobility was reduced or absent.
3. AOM Prone Children
[0196] An AOM prone child is a child having three episodes of AOM
within 6 months or 4 episodes within one year were considered
otitis prone while others who had fewer episodes were placed into
the non-otitis prone group.
4. Antibiotic
[0197] "Antibiotic" or like words or other forms refers to a
compound, substance, molecule, or composition, which acts to
reduce, inhibit, or prevent an infection of a bacteria.
5. Assaying
[0198] Assaying, assay, or like terms refers to an analysis to
determine a characteristic of a substance, such as a molecule or a
cell, such as for example, the presence, absence, quantity, extent,
kinetics, dynamics, or binding.
6. Assay Output
[0199] An "assay output" or like terms or other forms refers to the
result or product from running an assay, such as data. For example,
an assay output could be the fact that antibodies to P6 are present
in a sample, after running the assay which tested whether anti-P6
antibodies were present or not. The assay can be expressed in a
readout on a screen, on a paper, or in any other media, such as a
computer disk etc., but it must be expressed. In other words, the
fact of anti-P6 antibody presence is not the assay output, it is
the expression of this fact in some tangible form that is the assay
output.
7. Biological Sample/Sample
[0200] As used herein, the term "biological sample" or "sample"
refers to any material or substance from an individual or patient
that contains immune cells or antibodies. For example, the sample
can be blood, serum, urine or any other type of fluid.
8. Cell
[0201] The term "cell" as used herein also refers to individual
cells, cell lines, or cultures derived from such cells. A "culture"
refers to a composition comprising isolated cells of the same or a
different type. The term co-culture is used to designate when more
than one type of cell are cultured together in the same dish with
either full or partial contact with each other.
9. Comprise
[0202] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
10. Complex
[0203] The term complex as used herein refers to the association of
a first molecule with an another molecule for which the first
molecule has a binding affinity.
11. Components
[0204] Disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves to be
used within the methods disclosed herein. These and other materials
are disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. Thus, if a class of molecules A, B, and C are
disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited each is individually and
collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,
B-F, C-D, C-E, and C--F are considered disclosed. Likewise, any
subset or combination of these is also disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E would be considered
disclosed. This concept applies to all aspects of this application
including, but not limited to, steps in methods of making and using
the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed it is understood that each
of these additional steps can be performed with any specific
embodiment or combination of embodiments of the disclosed
methods.
12. Contacting
[0205] Contacting or like terms means bringing into proximity such
that a molecular interaction can take place, if a molecular
interaction is possible between at least two things, such as
molecules, cells, markers, at least a compound or composition, or
at least two compositions, or any of these with an article(s) or
with a machine. For example, contacting refers to bringing at least
two compositions, molecules, articles, or things into contact, i.e.
such that they are in proximity to mix or touch. For example,
having a solution of composition A and cultured cell B and pouring
solution of composition A over cultured cell B would be bringing
solution of composition A in contact with cell culture B.
[0206] It is understood that anything disclosed herein can be
brought into contact with anything else. For example, a sample can
be brought into contact with a reagent, such as an antibody that
binds P6, protein D and so forth.
13. Coapplication
[0207] "Coapplication" is defined as the application of one or more
substances simultaneously, such as in the same formulation or
consecutively, within a time frame such that each substance is
active during a point when the other substance or substances are
active.
14. Compounds and Compositions
[0208] Compounds and compositions have their standard meaning in
the art. It is understood that wherever, a particular designation,
such as a molecule, substance, marker, cell, or reagent
compositions comprising, consisting of, and consisting essentially
of these designations are disclosed. Thus, where the particular
designation marker is used, it is understood that also disclosed
would be compositions comprising that marker, consisting of that
marker, or consisting essentially of that marker. Where appropriate
wherever a particular designation is made, it is understood that
the compound of that designation is also disclosed. For example, if
particular biological material, such as EGF, is disclosed EGF in
its compound form is also disclosed.
15. Control
[0209] The terms control or "control levels" or "control cells" or
like terms are defined as the standard by which a change is
measured, for example, the controls are not subjected to the
experiment, but are instead subjected to a defined set of
parameters, or the controls are based on pre- or post-treatment
levels. They can either be run in parallel with or before or after
a test run, or they can be a pre-determined standard. For example,
a control can refer to the results from an experiment in which the
subjects or objects or reagents etc are treated as in a parallel
experiment except for omission of the procedure or agent or
variable etc under test and which is used as a standard of
comparison in judging experimental effects. Thus, the control can
be used to determine the effects related to the procedure or agent
or variable etc. For example, if the effect of a test molecule on a
cell was in question, one could a) simply record the
characteristics of the cell in the presence of the molecule, b)
perform a and then also record the effects of adding a control
molecule with a known activity or lack of activity, or a control
composition (e.g., the assay buffer solution (the vehicle)) and
then compare effects of the test molecule to the control. In
certain circumstances once a control is performed the control can
be used as a standard, in which the control experiment does not
have to be performed again and in other circumstances the control
experiment should be run in parallel each time a comparison will be
made.
16. Consisting Essentially of
[0210] "Consisting essentially of" in embodiments refers to, for
example, a surface composition, a method of making or using a
surface composition, formulation, or composition on the surface of
the biosensor, and articles, devices, or apparatus of the
disclosure, and can include the components or steps listed in the
claim, plus other components or steps that do not materially affect
the basic and novel properties of the compositions, articles,
apparatus, and methods of making and use of the disclosure, such as
particular reactants, particular additives or ingredients, a
particular agents, a particular cell or cell line, a particular
surface modifier or condition, a particular ligand candidate, or
like structure, material, or process variable selected. Items that
may materially affect the basic properties of the components or
steps of the disclosure or may impart undesirable characteristics
to the present disclosure include, for example, decreased affinity
of the cell for the biosensor surface, aberrant affinity of a
stimulus for a cell surface receptor or for an intracellular
receptor, anomalous or contrary cell activity in response to a
ligand candidate or like stimulus, and like characteristics.
17. Comparing
[0211] "Comparing" or like words or other forms refers to the act
of reviewing something in relation to something else.
18. Determining
[0212] "Determining" or like words or other forms refers to the act
of settling or deciding by choice from different alternatives or
possibilities.
19. Different Expression
[0213] The terms different expression and like terms can include
any difference including at least a 1%, 5%, 10%, 15%, 20%, 30%,
40%, 50%, 75%, 100%, 300%, 500%, 750%, 1000%, 5000%, 10,000%, or
50,000% difference.
20. Ear Infection
[0214] A middle ear infection (acute otitis media) is characterized
by inflammation of the ear drum (tympanic membrane) and the
accumulation of fluid behind the ear drum within the middle ear
space. Typically the middle ear fluid during AOM contains
inflammatory and immune cells and immune cell products and either
bacteria or viruses or both.
21. Epitope
[0215] As used herein, "epitope" refers to the region or fragment
of an antigen that can be recognized by the immune system.
Preferably, the epitope is recognized by antibodies, B cells or T
cells. An epitope can be linear or conformational.
[0216] There are 3 commonly used monoclonal antibodies for P6: 3B9,
7F3 and 4G4. Apicella and coworkers mapped the P6 conformational
epitope for antibody 3B9 using small peptides and competition
binding experiments. The results of Apicella's experiments pointed
to a non-continuous string of amino acids in the P6 sequence as
being important for 3B9 binding: GNTDERGT . . . RR (residues 87-94
and 147-148). (Bogdan J A, Apicella M A (1995) Mapping of a
surface-exposed, conformational epitope of the P6 protein of
Haemophilus influenzae. Infect Immun 63: 4395-4401.) Murphy and
coworkers proposed that the aspartate at position 59 is implicated
in antibody 7F3 binding to P6 (Murphy T F, Kirkham C, Sikkema D J
(1992) Neonatal, urogenital isolates of biotype 4 nontypeable
Haemophilus influenzae express a variant P6 outer membrane protein
molecule. Infect Immun 60: 2016-22). It has been confirmed that D59
was indeed part of the epitope to monoclonal antibody 7F3 using
nuclear magnetic resonance spectroscopy and ELISA. (Michel L V,
Kalmeta B, McCreary M, Snyder J, Craig P, Pichichero M E (2011)
Vaccine candidate P6 of nontypable Haemophilus influenzae is not an
outer membrane protein based on protein structural analysis,
Vaccine 29: 1624-1627) It has also been shown that monoclonal
antibody 4G4 competes for a similar epitope as 7F3 (Murphy T F,
Kirkham C, Sikkema D J (1992) Neonatal, urogenital isolates of
biotype 4 nontypeable Haemophilus influenzae express a variant P6
outer membrane protein molecule. Infect Immun 60: 2016-22 Bogdan J
A, Apicella M A (1995) Mapping of a surface-exposed, conformational
epitope of the P6 protein of Haemophilus influenzae. Infect Immun
63: 4395-4401), but no specific amino acids have been implicated in
4G4 binding. Additionally, the structure of P6 has been solved by
nuclear magnetic resonance spectroscopy [Parsons L M, Lin F, Orban
J. Peptidoglycan recognition by Pal, an outer membrane lipoprotein.
Biochemistry 2006; 45: 2122-28].
22. Higher
[0217] The terms "higher," "increases," "elevates," or "elevation"
or variants of these terms, refer to increases above basal levels,
e.g., as compared to a control. The terms "low," "lower,"
"reduces," or "reduction" or variation of these terms, refer to
decreases below basal levels, e.g., as compared to a control. For
example, basal levels are normal in vivo levels prior to, or in the
absence of, or addition of an agent such as an agonist or
antagonist to activity.
23. Immune Response
[0218] "Immune response" is how the body recognizes and defends
itself against foreign substances such as bacteria, viruses,
toxins, drugs, etc. An immune response can also be the body's
response to substances that appear to be foreign even if the
substances are actually self proteins.
24. In Vitro In Vivo
[0219] The terms in vitro and in vivo as used herein have their
usual and ordinary meanings in the art.
25. Inhibit
[0220] By "inhibit" or other forms of inhibit means to hinder or
restrain a particular characteristic. It is understood that this is
typically in relation to some standard or expected value, in other
words it is relative, but that it is not always necessary for the
standard or relative value to be referred to. For example,
"inhibits phosphorylation" means hindering or restraining the
amount of phosphorylation that takes place relative to a standard
or a control.
26. Infection
[0221] Infections of the human host are caused by bacteria,
viruses, fungi and parasites. Infections elicit an inflammatory and
immune response by the human host to eliminate the organism
27. Lung Infection
[0222] Lung infections may be caused by bacteria, viruses, fungi
and parasites and the pathological process is confined to the lower
airways consisting of the trachea, bronchi, bronchioles and lung
parenchyma.
28. Material
[0223] Material is the tangible part of something (chemical,
biochemical, biological, or mixed) that goes into the makeup of a
physical object.
29. Molecule
[0224] As used herein, the terms "molecule" or like terms refers to
a biological or biochemical or chemical entity that exists in the
form of a chemical molecule or molecule with a definite molecular
weight. A molecule or like terms is a chemical, biochemical or
biological molecule, regardless of its size.
[0225] Many molecules are of the type referred to as organic
molecules (molecules containing carbon atoms, among others,
connected by covalent bonds), although some molecules do not
contain carbon (including simple molecular gases such as molecular
oxygen and more complex molecules such as some sulfur-based
polymers). The general term "molecule" includes numerous
descriptive classes or groups of molecules, such as proteins,
nucleic acids, carbohydrates, steroids, organic pharmaceuticals,
small molecule, receptors, antibodies, and lipids. When
appropriate, one or more of these more descriptive terms (many of
which, such as "protein," themselves describe overlapping groups of
molecules) will be used herein because of application of the method
to a subgroup of molecules, without detracting from the intent to
have such molecules be representative of both the general class
"molecules" and the named subclass, such as proteins. Unless
specifically indicated, the word "molecule" would include the
specific molecule and salts thereof, such as pharmaceutically
acceptable salts. t is understood that molecules can include
recombinant variations or humanized variations or oligomeric or
non-oligomeric variations where approriate.
30. Normalizing
[0226] Normalizing or like terms means, adjusting data, or a
response, or an assay result, for example, to remove at least one
common variable.
31. Optionally
[0227] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
32. Obtaining
[0228] "Obtaining" or like words or other forms refers to getting
or receiving or attaining. It requires to a planned effort by the
actor, but the plan can be in acceptance, for example, by accepting
something that is given one.
33. Pharmacological Activity
[0229] As used herein, the term "pharmacological activity" refers
to the inherent physical properties of a peptide or polypeptide.
These properties include but are not limited to half-life,
solubility, and stability and other pharmacokinetic properties.
34. Pneumonia
[0230] Pneumonia is an infection of the lung parenchyma. If the
infection involves the bronchi it is often termed bronchitis or
bronchopneumonia.
[0231] Streptococcus pneumonia is a bacteria that causes ear
infections, sinus infections, bronchopneumonia, pneumonia,
bacteremia, septicemia, meningitis, and other
bloostream-disseminated infections such as arthritis.
35. Positive Control
[0232] A "positive control" or like terms is a control that shows
that the conditions for data collection can lead to data
collection.
36. Prevent
[0233] By "prevent" or other forms of prevent means to stop a
particular characteristic or condition. Prevent does not require
comparison to a control as it is typically more absolute than, for
example, reduce or inhibit. As used herein, something could be
reduced but not inhibited or prevented, but something that is
reduced could also be inhibited or prevented. It is understood that
where reduce, inhibit or prevent are used, unless specifically
indicated otherwise, the use of the other two words is also
expressly disclosed. Thus, if inhibits phosphorylation is
disclosed, then reduces and prevents phosphorylation are also
disclosed.
37. Prescribing, Prescription
[0234] "Prescribing" or "Prescription" or like words or other forms
refers to a written direction or act for a therapeutic or
corrective agent; specifically, such as one for the preparation and
use of a medication.
38. Primers
[0235] "Primers" are a subset of probes which are capable of
supporting some type of enzymatic manipulation and which can
hybridize with a target nucleic acid such that the enzymatic
manipulation can occur. A primer can be made from any combination
of nucleotides or nucleotide derivatives or analogs available in
the art, which do not interfere with the enzymatic
manipulation.
39. Probes
[0236] "Probes" are molecules capable of interacting with a target
nucleic acid, typically in a sequence specific manner, for example
through hybridization. The hybridization of nucleic acids is well
understood in the art and discussed herein. Typically a probe can
be made from any combination of nucleotides or nucleotide
derivatives or analogs available in the art.
40. Pro-Drug
[0237] The term "pro-drug or prodrug" is intended to encompass
compounds which, under physiologic conditions, are converted into
therapeutically active agents. A common method for making a prodrug
is to include selected moieties which are hydrolyzed under
physiologic conditions to reveal the desired molecule. In other
embodiments, the prodrug is converted by an enzymatic activity of
the host animal.
41. Ranges
[0238] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data are provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular datum point "10" and a particular
datum point 15 are disclosed, it is understood that greater than,
greater than or equal to, less than, less than or equal to, and
equal to 10 and 15 are considered disclosed as well as between 10
and 15. It is also understood that each unit between two particular
units are also disclosed. For example, if 10 and 15 are disclosed,
then 11, 12, 13, and 14 are also disclosed.
42. Reduce
[0239] By "reduce" or other forms of reduce means lowering of an
event or characteristic. It is understood that this is typically in
relation to some standard or expected value, in other words it is
relative, but that it is not always necessary for the standard or
relative value to be referred to. For example, "reduces
phosphorylation" means lowering the amount of phosphorylation that
takes place relative to a standard or a control.
43. References
[0240] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
44. RT, PCR, qPCR
[0241] "RT, PCT, and qPCR" refer to molecular biology techniques,
Reverse Transcriptase, Polymerase Chain Reaction, and quantitative
PCR respectively. These techniques allow for the detection and
amplification of nucleic acids from cells.
45. Sinus Infection
[0242] Sinus infections are commonly termed sinusitis or
rhinosinusitis. Inflammation occurs in the sinus spaces, consisting
of the maxillary, ethmoid, frontal and sphenoid sinuses.
46. Subject
[0243] As used throughout, by a "subject" is meant an individual. A
subject can be a patient. A subject can be preferably less than 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.
47. Standard
[0244] A "standard" or like terms or other forms refers to an
established rule or measure that has been previously determined,
but which can be used for comparative purposes. It often is used
like a control, and often it is produced by running a control or
multiple control experiments to determine a consistent or average
result as a "control."
48. Substance
[0245] A substance or like terms is any physical object. A material
is a substance. Molecules, cells, proteins, and DNA can be
considered substances. A machine or an article would be considered
to be made of substances, rather than considered a substance
themselves.
49. Tissue
[0246] Tissue or like terms refers to a collection of cells.
Typically a tissue is obtained from a subject.
50. Transmitting the Assay Output to a Recipient
[0247] "Transmitting the assay output to a recipient" or like terms
or other forms refers to the act of sending an assay output. This
can refer to for example, refer to an email from a computer,
automatically generated to, for example, a doctor or doctor's
office.
51. Treating
[0248] "Treating" or "treatment" does not mean a complete cure. It
means that the symptoms of the underlying disease are reduced,
and/or that one or more of the underlying cellular, physiological,
or biochemical causes or mechanisms causing the symptoms are
reduced. It is understood that reduced, as used in this context,
means relative to the state of the disease, including the molecular
state of the disease, not just the physiological state of the
disease. In certain situations a treatment can inadvertantly cause
harm.
52. Treatment
[0249] By "treatment" and "treating" is meant the medical
management of a subject with the intent to cure, ameliorate,
stabilize, or prevent one or more symptoms of a disease,
pathological condition, or disorder. This term includes active
treatment, that is, treatment directed specifically toward the
improvement of a disease, pathological condition, or disorder, and
also includes causal treatment, that is, treatment directed toward
removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes
prophylactic or palliative treatment, that is, treatment designed
for the relief of symptoms rather than the curing of the disease,
pathological condition, or disorder; preventative treatment, that
is, treatment directed to minimizing or partially or completely
inhibiting the development of the associated disease, pathological
condition, or disorder; and supportive treatment, that is,
treatment employed to supplement another specific therapy directed
toward the improvement of the associated disease, pathological
condition, or disorder. It is understood that treatment, while
intended to cure, ameliorate, stabilize, or prevent a disease,
pathological condition, or disorder, need not actually result in
the cure, ameliorization, stabilization or prevention. The effects
of treatment can be measured or assessed as described herein and as
known in the art as is suitable for the disease, pathological
condition, or disorder involved. Such measurements and assessments
can be made in qualitative and/or quantitative terms. Thus, for
example, characteristics or features of a disease, pathological
condition, or disorder and/or symptoms of a disease, pathological
condition, or disorder can be reduced to any effect or to any
amount.
53. Therapeutically Effective
[0250] The term "therapeutically effective" means that the amount
of the composition used is of sufficient quantity to ameliorate one
or more causes or symptoms of a disease or disorder. Such
amelioration only requires a reduction or alteration or decrease,
not necessarily elimination. The term "carrier" means a compound,
composition, substance, or structure that, when in combination with
a compound or composition, aids or facilitates preparation,
storage, administration, delivery, effectiveness, selectivity, or
any other feature of the compound or composition for its intended
use or purpose. For example, a carrier can be selected to minimize
any degradation of the active ingredient and to minimize any
adverse side effects in the subject.
[0251] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
EXAMPLES
A. Example 1
Bactericidal Antibody Response Against P6, Protein D and OMP26 of
Non Typeable Haemophilus influenzae (NTHi) after Acute Otitis Media
(AOM) in Otitis Prone Children
1. Background
[0252] NontypeableHaemophilus influenzae (NTHi) is one of the major
causes of infections in the upper respiratory tract and middle ear
leading to AOM. Bactericidal antibody provides protection from AOM
caused by NTHi. Understanding the contributions of bactericidal
antibody specific to potential vaccine antigens will help in the
design of a novel vaccine, which could protect against NTHi
infections.
[0253] Protein D, P6, and OMP26 are three conserved outer membrane
proteins of NTHi currently being considered as vaccine candidates
against infections caused by NTHi.
[0254] The aim of the present study was to investigate the antigen
specific bactericidal antibody response, to protein D, P6 and OMP26
in otitis prone children.
2. Methods
[0255] i. Study Design
[0256] The patients constituted a consecutively studied series of
27 children; aged 7 months to 28 months of age (mean
15.41.+-.5.7mo) who were otitis prone between the age of 6 and 36
months. From these 27 children we had 16 acute sera collected at
the time of diagnosis of AOM caused by NTHi and 26 serum samples
from the convalescent stage.
ii. Tympanocentesis
[0257] All diagnoses of AOM for the defining event that caused the
child to meet the definition of otitis prone was confirmed by
tympanocentesis. Middle ear fluid (MEF) for culture was obtained by
puncture of the inferior portion of an intact TM with a 20-gauge
spinal needle attached to a 3-mL syringe using a hand-held
operating otoscope. If a small sample of MEF was obtained on
aspiration, 0.5 ml of trypticase soy broth was aspirated through
the spinal needle and then aliquoted and inoculated onto agar
plates and into broth, as described below.
iii. Bacteriology
[0258] Middle ear fluid were cultured on chocolate agar plates and
inoculated in BHI with 15% glycerol and preserved at -80 degree.
The NTHi strains were identified by standard laboratory procedures.
An isolate was identified as NTHi based on colony morphology,
porphyrin reactivity, and growth requirement for hemin and
nicotinamide adenine dinucleotide using Haemophilus ID Quad plates
[McCrea et al., J. Clin. Microbiol. 2008; 46: 406-416].
iv. Purification of P6, OMP26 and Protein D
[0259] Recombinant protein D was obtained from GlaxoSmithKline
(GSK, Rixensart, Belgium). The P6 plasmid was obtained from Tim
Murphy, University of Buffalo and the OMP26 plasmid was obtained
from Jennelle Kyd, University of Can berra, Australia. P6 and OMP26
were expressed in E coli BL21 (DE3). P6 was expressed predominantly
as inclusion bodies and purified under denaturing conditions. OMP26
was expressed in soluble fraction and purified under native
conditions. The purification was carried out with an already
published procedure [Adhami et al., Infection and Immunity 1999;
67: 1935-1942; Bhushan et al., Infection and Immunity 1997; 65:
2668-75]. The purity of purified recombinant proteins was assessed
by SDS-PAGE.
v. Bactericidal assay
[0260] Forty two serum samples were screen for bactericidal
activity against their homologous NTHi strain (isolated from MEF of
that child). 21 bactericidal serum samples were selected to assess
bactericidal activity against a heterologous strain (86-028 NP
provided as a gift from Lauren Bakaletz, Ohio State University,
Columbus, Ohio). The sera were heat-inactivated at 56.degree. C.
for 30 minutes to eliminate human complement. Homologous and
heterologous NTHi strains were cultivated, harvested, and diluted
to a concentration of .about.10.sup.5 CFU/ml. Twelve serial twofold
dilutions of the serum to be tested (starting at 1:2) were mixed
with precolostral calf serum complement (PCCS) and 20 .mu.l of
bacteria. After 60 minutes of incubation, the number of surviving
bacteria was determined by plating 5 .mu.l onto chocolate agar and
counting the colonies. The bactericidal titer of the serum was
defined as the inverse of the highest dilution that led to
.gtoreq.50% bacterial killing and was compared to that of negative
control (complement plus bacterium). In house developed appropriate
controls were included in all experiments.
vi. Adsorption of Anti P6, Protein D and OMP26 Antibodies from
Sera
[0261] The bactericidal sera were later depleted for anti protein
D, P6, and OMP26 specific antibodies and used for bactericidal
assay. For the absorption procedure, polystyrene beads were washed
extensively with borate buffer (pH 8.5) and resuspended in 1 ml of
Borate buffer. Recombinant protein D, P6 and OMP26 antigens were
incubated with these beads overnight at room temperature. The beads
were washed extensively, incubated in BSA/Borate buffer for 30
minutes at room temperature, then pelleted and incubated with 200
.mu.l of patient sera for 2 hours at room temperature. The beads
were centrifuged (200.times.g) and the supernatant was collected.
The anti protein D, P6 and OMP26 absorbed sera levels were measured
by ELISA (below mentioned) and used for bactericidal assays. The
reciprocal bactericidal titers were compared with unadsorbed sera
to determine the bactericidal activity mediated by each of the
specific antibodies.
vii. Detection of P6, Protein D and OMP26 Specific IgG by ELISA
[0262] Protein D, P6 and OMP26 specific IgG antibody titers in the
acute and convalescent serum samples were determined by ELISA.
Protein D, P6 and OMP26 recombinant proteins were coated on 96 well
plate with the concentration of 0.25 ug/ml each in coating buffer.
After blocking with 3% skim milk, diluted serum samples were added
to the wells, and the mixture was incubated at room temperature for
1 h. affinity purified goat anti human IgG antibody conjugated to
horseradish-peroxidase was used as a secondary antibody. The
reaction products were developed with TMB Microwell peroxidase
substrate system, stopped by the addition of 1.0 M phosphoric acid,
and read by ELISA reader at 450 nm.
viii. Detection of Whole Cell NTHi IgG Antibodies by ELISA
[0263] For the whole cell specific ELISA, for each child their
homologous NTHi strain isolated from MEF and a heterologous strain
(86-028 NP) were used. Homologous and heterologous strains were
grown on chocolate agar and further inoculated into brain heart
infusion broth supplemented with NAD and Hemin. The bacteria were
grown to mid log phase, harvested, and washed with PBS containing
0.15 mM CaCl2, and 0.5 mM MgCl2. The pellet was finally suspended
and diluted to an OD of 1 at 490 nm. The NTHi preparation was used
to coat 96-well plates. After blocking with 1% gelatin and washing,
diluted serum was added to the wells, and the mixture was further
incubated at room temperature for 1 h. Affinity purified goat anti
human IgG antibody conjugated to alkaline phosphatase was used as a
secondary antibody. The reaction products were developed with PNP
dissolved in diethanolamine buffer. The reaction was stopped by the
addition of 2M NaOH and was read by ELISA reader (molecular
devices) at 405 nm. Titers for test samples were determined
relative to a reference serum run on the same plate, and values
were expressed relative to reference serum.
ix. Statistics
[0264] Student t test was used to analyze the significance of
antibody changes. P value<0.05 was considered as significant.
Statistical analysis of correlation coefficients by linear
regression (r.sup.2) between ELISA titers and bactericidal titers
was determined measure the level of correlation between the
assays.
3. Results
[0265] The bactericidal antibody response to 3 nontypeable
Haemophilus influenzae (NTHi) outer membrane proteins (Protein D,
P6 and OMP26) was studied in 27 otitis prone children (age 7-28
months) after an acute otitis media (AOM) caused by NTHi. Among 17
acute serum samples, only 4 sera (24%) had detectable bactericidal
activity versus 18 of 25 convalescent serum samples (72%). 11 sera
(58%) had bactericidal activity against a heterologous NTHi strain
but the titers were significantly lower (p=0.0023) as compared to
the homologous strains. Levels of protein D (p 0.002) (FIG. 1C) and
P6 (p=0.003) (FIG. 1B) but not OMP26 antibodies were higher in
bactericidal sera compared to non bactericidal sera.
[0266] Serum IgG antibody levels to whole cell homologous and
heterologous NTHi strains were measured and compared in
bactericidal and non-bactericidal serum samples. FIG. 1D
illustrates that the 95% confidence interval was 768 to 1994 and
whole cell IgG titers were significantly higher in bactericidal
sera as compared to non-bactericidal sera (p=0.001). The 95%
confidence interval between the whole cell ELISA for homologous and
heterologous strains was 199 to 1421 and titers were significantly
higher for homologous NTHi strains as compared to the heterologous
strain (p=0.03) (FIG. 2A).
[0267] Serum IgG titers (protein D and P6) and bactericidal titers
correlated (r=0.44 for protein D and 0.48 for P6; p=0.07 and 0.002,
respectively) (FIGS. 2B and 2C). For 3 (14%) and 18 (86%) of 21
bactericidal sera tested, removal of anti-protein D and P6
antibody, respectively resulted in a significant drop in
bactericidal antibody (p<0.005 for P6) (Table 1). Two children
showed 25% bactericidal activity directed towards protein D and one
child showed 50% bactericidal activity directed to protein D. For
18 (86%) of the same 21 bactericidal sera tested, a significant
drop in bactericidal activity was measured after removal of P6
antibody from sera (p<0.005). P6 specific bactericidal antibody
in the 18 serum samples accounted for almost 50% of the total
bactericidal activity measured. OMP26 was not observed to
contribute in bactericidal activity as the removal of OMP26
antibody did not change the bactericidal titers (data not
shown).
TABLE-US-00004 TABLE 1 Representation of the relative contribution
of anti P6 and protein D antibodies in total bactericidal activity
in bactericidal serum samples Bactericidal Bactericidal
Bactericidal titers Titers Pre titers post post adsorption Patient
No adsorption adsorption (P6) (protein D) 06-01-041(AOM1 F/U) 8 4 6
06-01-023 (AOM1 F/U) 16 4 16 07-01-061(AOM1 F/U) 128 64 128
06-01-006 (AOM1) 64 32 64 07-01-062 (AOM1) 4 4 4 07-01-062 (AOM1
F/U) 32 16 24 08-01-078 (AOM2) 8 4 4 08-01-78 (AOM2 F/U) 64 32 64
06-01-037 (AOM1 F/U) 8 4 8 07-01-051 (AOM1 F/U 64 16 64 06-01-006
(AOM1 F/U) 8 4 8 06-01-031 (AOM1 F/U) 64 32 64 06-01-021 (AOM1 F/U)
64 16 64 06-01-035 (AOM1 F/U) 16 8 16 06-01-043 (AOM1 F/U) 128 128
128 09-03-085 (AOM2 F/U) 8 4 8 01-072 (AOM2 F/U) 128 32 128 03-066
(AOM2 F/U) 128 64 128 01-038 (AOM1 F/U) 128 64 128 01-033 (AOM1
F/U) 256 128 256 09-01-093 (AOM1 F/U 4 4 4
[0268] Anti protein D, P6, and OMP26 specific antibodies were
selectively depleted from 21 bactericidal sera in order to assess
their relative contribution in total bactericidal antibody titers.
After absorption all sera were tested for residual antibody to
protein D, P6, and OMP26 by ELISA to demonstrate the absorption was
complete (data not shown). Cross adsorption experiments were
performed to demonstrate that the adsorption was quite specific.
For instance, P6 and OMP26 ELISA titers were quantified in sera
which were adsorbed for Protein D antibodies and results showed
that there was no cross adsorption. Each adsorption experiment was
carried out on two different occasions to look at the
reproducibility of results and ELISA titers were established after
every adsorption.
4. Conclusions
[0269] In this study, the bactericidal antibody response was
measured against three vaccine candidate proteins of NTHi--protein
D, P6, and OMP26--in otitis prone children. This is the first study
to assess the proportional contribution of serum bactericidal
activity against these three conserved outer membrane proteins of
NTHi. These data indicate several important findings: 1.
Bactericidal antibody in otitis prone children is infrequently
present in acute sera but present in the majority of children in
convalescence 2. The bactericidal antibody that otitis prone
children develop is primarily but not exclusively to the homologous
infecting strain. 3. Total antibody to protein D and P6 partially
correlate with bactericidal antibody 4. Protein D does not elicit
bactericidal antibody in a majority of otitis prone children. 5. P6
does elicit bactericidal antibody in a majority of otitis prone
children. 6. OMP26 elicits no detectable bactericidal antibody in
otitis prone children. 7. A variable proportion of serum
bactericidal antibody is not directed against protein D or P6 and
must be directed to other unconserved or conserved outer membrane
proteins.
[0270] Shurin et al, (1980) suggested that bactericidal activity in
sera to the homologous strain develops during the convalescent
stage of non otitis prone children [Shurin et al., The Journal of
Paediatrics 1980; 97: 364-369]. Later, Yamanaka and Faden (1992)
showed that acute sera infrequently had bactericidal antibodies to
homologous NTHi following AOM [Yamanaka and Faden, The Journal of
Pediatrics 1992; 122: 212-218]. Faden et al (1989) reported that
the bactericidal antibody response is NTHi strain specific and
provided little or no cross protection in otitis prone and non
otitis prone children with otitis media [Faden et al., The Journal
of Infectious Disease 1989; 160: 999-1004]. Bernstein et al (1991)
found in three otitis prone children that the bactericidal antibody
response was not cross protective against a heterologous strain of
NTHi causing a second or third episode [Bernstein et al.,
Otolaryngology--Head and Neck Surgery 1991; 105: 406-410]. The
findings of the current data are in agreement with some aspects of
previous work in regards to bactericidal antibody directed to the
homologous strain prior to and after an NTHi AOM. However,
bactericidal activity against a heterologous strain was not found
in otitis prone children, albeit at lower titers as compared to the
homologous strain. In the present study, the detection of
bactericidal antibody titers in otitis prone children argues
against the notion that these children are incapable of generating
immune responses to NTHi as an otopathogen. However the differences
between homologous and heterologous strain bactericidal activity
suggests a probable reason of failure to protect against
heterologous new infections and points to the necessity of a
multi-component NTHi vaccine.
[0271] The focus of the comparative analysis of bactericidal
antibody and antibody class was centered on IgG. The IgM responses
were not taken into consideration, as they are not predictive of
immunological memory or affinity maturation. Chen at al (1999)
demonstrated that serum IgG levels are higher in bactericidal sera
against Moraxella catarrhalis in healthy adults and children [Chen
et al., Infection and Immunity 1999; 67: 1310-1316]. IgG antibodies
to P6 have been found to be bactericidal in an experimental animal
system [De Maria et al., Infection and Immunity 1996; 64:
5187-5192] and to be associated, in part, with the bactericidal
activity in otitis prone children [Sabirov et al., Paediatric
Research 2009; 66: 565-570]. Consistent with those previous
findings and Yamanaka and Faden in otitis prone children [Yamanaka
and Faden, The Journal of Pediatrics 1992; 122: 212-218], higher
IgG anti P6 levels were found in bactericidal sera of otitis prone
children.
[0272] A modest correlation was found between bactericidal titers
and serum IgG antibody concentrations for protein D and P6 but not
OMP26. It is not fully understood whether the lack of a stronger
correlation is related to a disparity in antibody avidity as
detected in the two assays. In the past also, high IgG antibody
levels against P6 with no commensurate bactericidal activity has
been attributed to IgG antibody of affinity and/or, antibodies
directed against certain non-critical epitopes [Yamanaka and Faden,
The Journal of Pediatrics 1992; 122: 212-218]. Avidity of antibody
in children improves with age and immune maturation [Pollard et
al., The Lancet 2000; 356: 2065-66; Maslanka et al., Infection and
Immunity 1998; 66: 2453-59]; this area needs further investigation.
No correlation was found between OMP26 IgG levels and bactericidal
titers (data not shown) and OMP26, consistent with one previous
report [Cripps and Otczyk, Exp Rev 685 Vaccines 2006;
5:517-34].
[0273] Significantly higher whole cell ELISA titers were found in
bactericidal sera as compared to non bactericidal sera. The IgG
titers were significantly higher for the homologous strain as
compared to the heterologous strain. The results were quite
comparable with the serum IgG titers for P6 and protein D in
bactericidal and non bactericidal sera. There is limited literature
on the relationship of whole cell ELISA titers and bactericidal
titers. The present study indicates that bactericidal sera would
have higher IgG titers against the homologous strain than non
bactericidal sera and heterologous NTHi strains.
[0274] This study is the first to examine the exact proportion of
bactericidal antibody directed against protein D, P6 and OMP 26 in
otitis prone children after AOM. Forsgren has previously shown that
antibody to protein D was bactericidal in rat model [Kyd and
Cripps, Infect Immun 1998; 66: 2272-2278; Forsgreen et al.,
Clinical Infectious Disease 2008; 46: 726-31]. The role of protein
D in bactericidal activity in children following AOM has not been
previously studied. The results indicate that protein D specific
antibodies contribute to bactericidal activity in about 25% of
otitis prone children. In contrast the contribution of anti-P6
antibody to bactericidal activity in over 80% of otitis prone
children is noteworthy, especially since the anti-P6 antibody
appeared to contribute about 50% of the total bactericidal activity
in many sera.
[0275] Among the 21 bactericidal sera, the proportion of
bactericidal antibody response to three conserved outer membrane
antigenic determinants did not exceed more than 50% in 18 sera.
This result indicates that a significant percentage of bactericidal
antibodies are directed to other proteins. The structurally
non-conserved porins are one of the classes of proteins on the NTHi
surface that have been shown to elicit bactericidal antibodies
[Neary et al., Infection and Immunity 2001; 69:773-778; Sikkema and
Murphy, Infection and Immunity 1992; 60:5204-5211]. Nevertheless,
there might be other yet unidentified, conserved targets for
bactericidal antibodies to NTHi.
[0276] The children above 6 months were deliberately taken for the
study to have the minimal effect mediated by the maternal
antibodies in the serum [Zinkernagel, The New England Journal of
Medicine 2001; 345:1331-1335]. Therefore it was assumed that the
bactericidal activity detected in the otitis prone children is
totally conferred by their own immune system and not by passive
transfer from mother.
[0277] In summary, the findings establish that a significant
portion of bactericidal activity is directed to OMP P6 and to a
lesser degree to protein D in otitis prone children following a
naturally acquired AOM infection by NTHi. The contribution of other
NTHi conserved antigens displayed on the NTHi surface is encouraged
by the results herein. The lack of a strong correlation between
bactericidal antibody titers and IgG titers indicates the need to
establish a biologically relevant serologic surrogate to define a
particular bactericidal titer, a threshold for defining a
protective titer, which is critical for vaccine development.
B. Example 2
T Cells and AOM
1. Introduction
[0278] Streptococcus pneumoniae (Spn) and non-typeable Haemophilus
influenzae (NTHi) are the two most common pathogens causing AOM
[Casey et al., Pediatr. Infect. Dis. J. 2010; 29(4):304-9]. In
animal models, CD4.sup.+ T lymphocytes have been shown to be
critical for protective immunity against these prevalent bacterial
respiratory pathogens [Malley et al., Proc. Natl. Acad. Sci. U.S.A
2005; 102(13):4848-53; McCool and Weiser, Infect. Immun. 2004;
72(10):5807-13; Snapper et al., Trends Immunol. 2001;
22(6):308-11]. More recently, Th-17 cells secreting IL-17, IL-21,
and IL-22 have been described to impart antibody independent
protection in mouse model of pneumococcal infection [Malley et al.,
Infect. Immun. 2006; 74(4):2187-95]. In older children (median age
5 years) and adults, antigen-specific CD4.sup.+ T-cells has been
shown to reduce Spn nasopharyngeal colonization [Mureithi et al.,
J. Infect. Dis. 2009; 200(5):783-93; Zhang et al., J. Infect. Dis.
2007; 195(8):1194-202]. An effective pathogen-specific T-cell
response in adults has been associated with protection from
invasive Spn disease (IPD) and chronic obstructive pulmonary
disease (COPD) caused by Spn and NTHi respectively [King et al.,
Am. J. Respir. Crit Care Med. 2003; 167(4):587-92; de Bree et al.,
J. Infect. Dis. 2007; 195(11):1718-25]. However, there are no data
that correlate a protective role of CD4.sup.+ T-helper subsets
among children experiencing AOM.
[0279] Robust memory T- and B-cell responses are generated during
both onset of a natural infection as well as upon vaccination, with
memory lymphocytes populating lymphoid and non-lymphoid sites [de
Bree et al., J. Infect. Dis. 2007; 195(11):1718-25; Lanzavecchia
and Sallusto, Curr. Opin. Immunol. 2009; 21(3):298-304; Kelly et
al., JAMA 2005; 294(23):3019-23]. Once generated, memory T-cells
and antibodies can be detected in the blood circulation over a
period of time [de Bree et al., J. Infect. Dis. 2007;
195(11):1718-25; Pitcher et al., Nat. Med. 1999; 5(5):518-25]. In
both humans and mice, CD4.sup.+ T-cells comprise functionally
distinct populations characterized by specific cytokine profiles
produced in response to antigens [Fietta and Delsante, Annu. Rev.
Immunol. 2009; 27:485-517; Korn et al., Annu. Rev. Immunol. 2009;
27:485-517]. More recently, follicular helper T (Tfh) cells have
been shown as a major subset to provide help to B-cells for
antibody responses [Fazilleau et al., Immunity. 2009; 30(3):324-35;
Yu and Vinuesa, Trends Immunol. 2010; 31(10):377-83; Morita et al.,
Immunity. 2011; 34(1):108-21].
[0280] To explain the immunological dysfunction that leads to
recurrent AOM, earlier studies have found lower levels of
otopathogen-specific antibody concentrations in otitis prone
children, as compared to non-otitis prone children [Faden, Eur. J.
Pediatr. 2001; 160(7):407-13; Pichichero et al., Vaccine 2010;
28:7184-7192]. This work provides a better understanding of the
immunologic dysfunction in otitis prone children, focusing on the
generation of different subsets (Th1, Th2 & Th-17) of memory
CD4.sup.+ T-helper cells in correlation with B-cell antibody
responses as a possible novel explanation. Using six pneumococcal
and three NTHi protein antigens, we enumerated Spn and
NTHi-specific functional CD4.sup.+ T-helper memory cell subsets in
the peripheral blood of a cohort of non-otitis prone and otitis
prone children. Serum IgG responses were also measured to the same
antigens in these children.
2. Methods
[0281] i. Subjects
[0282] Subjects were participants from a 5-year prospective
longitudinal AOM study funded by the NIH NIDCD [Pichichero et al.,
Vaccine 2010; 28:7184-7192]. Enrolled children were from a middle
class, suburban socio-demographic population in Rochester N.Y.
Healthy children at age of 6 months without prior AOM were enrolled
and had blood, nasopharyngeal (NP) and oropharyngeal (OP) cultures
obtained seven times, at the age of 6, 9, 12, 15, 18, 24 and 30
months. Middle ear fluid (MEF) was obtained by tympanocentesis
during AOM episodes. Colonization with Spn and/or NTHi in the NP/OP
and MEF was routinely determined by standard microbiologic culture.
To identify the otitis prone child in the study population all the
children had tympanocentesis-confirmed infections and all received
antibiotic therapy directed to the otopathogen isolated from middle
ear fluid for each AOM event. PBMCs were isolated from the
collected blood and frozen in the liquid nitrogen until used.
Children having three episodes of AOM within 6 months or 4 episodes
within one year were considered otitis prone while others who had
fewer episodes were placed into the non-otitis prone group. Written
informal consent was obtained in association with a protocol
approved by the Rochester General Hospital Investigational Review
Board.
ii. Antigens
[0283] Six different pneumococcal protein antigens were used in
this study: pneumococcal histidine triad proteins D (PhtD) and E
(PhtE), LytB, PcpA, PlyD1 (a detoxified derivative of pneumolysin
which has three point mutations that do not interfere with
anti-pneumolysin antibody responses) and PspA. Haemophilus
influenzae protein antigens used were P6, OMP26, and Protein D. An
optimal dosage for stimulation was determined by absence of
detectable cell toxicity, by the use of tryptan blue staining
and/or flow cytometry analysis after propidium iodide staining
(data not shown). Staphylococcal enterotoxin B (Sigma, St Louis)
was used as a positive control.
Iii. T Cell Stimulation
[0284] T cell stimulation and intracellular cytokine profiling was
standardized in our laboratory adapted from elsewhere [Lamoreaux et
al., Nat. Protoc. 2006; 1(3):1507-16]. Briefly, PBMCs were
stimulated with the six pneumococcal antigens or the three NTHi
antigens individually depending on the NP colonizing or AOM
causative pathogen. Prior to stimulation, frozen PBMCs were quickly
thawed in a 37.degree. C. water bath followed by slowly adding
complete culture medium (RPMI 1640 supplemented with 10% of FBS, 2
mM L-glutamine, 0.1 mM sodium pyruvate, nonessential amino acids,
100 U/mL penicillin, 100 .mu.g/mL streptomycin). Cells were then
washed and rested overnight in complete culture media in 24-well
plates. PBMCs were stimulated using a standardized protocol in our
laboratory. Briefly, cells were counted and 1.times.10.sup.6 cells
were placed in the each well of a 96-well flat bottom culture plate
for stimulation with either 1 .mu.g/ml of various protein antigens
individually or with 1 .mu.g/ml of Staphylococcal enterotoxin B
(SEB). Cells left untreated served as negative controls. Cells were
then incubated for 2 h at 37.degree. C. in the presence of 5%
CO.sub.2 for antigen processing. After 2 hours, Golgi transport
inhibitors (Brefeldin A and Monensis; BD Biosciences) were added to
preserve cytokines intracellularly and incubation was then
continued for an additional 4 hours. To the cells 1 .mu.g/ml
concentrations of anti-CD28 and anti-CD49d antibodies (clones L293
and L25 respectively; BD Biosciences) were added to provide
co-stimulation and enhance the detection of antigen specific
responses. Anti-CD28 and CD49d antibodies have been widely used for
co-stimulation without affecting background levels [Pitcher et al.,
Nat. Med. 1999; 5(5):518-25].
Iv. Cell Surface Staining and Cytokine Profiling
[0285] An intracellular cytokine staining assay (ICCS) was used to
evaluate antigen specific CD4.sup.+ T-cell subsets (Th-1, Th-2 and
Th-17). After stimulation, cells were transferred to 96-well
V-bottom plates and washed once with FACS buffer (PBS with 5% FBS)
and stained with the antibodies to various cell surface markers.
Antibodies used were anti-CD4 APC Alexafluor 750 (clone RPA T4,
eBiosciences), PE-Texas Red anti-CD45RA (clone MEM56, Invitrogen),
anti-CCR7PerCP/Cy5.5 conjugate (clone TG8/CCR7, Biolegend). To
identify Tfh cells in the PBMC samples, cells were surface stained
with anti-CXCR5 perCP cy5.5 (Biolegend) anti-CD4 APC Alexafluor
750, PE-Texas Red anti-CD45RA and anti-CD3 Qdot (clone UCHT1,
Invitrogen) separately. Cells were then permeabilized with fixation
and permeabilization solution (BD Biosciences) for 20-minutes and
washed three times with 1.times. permeabilization buffer (BD
Biosciences). A cocktail of various cytokine specific antibodies
was used to stain intracellularly captured cytokines as a result of
stimulation. Antibodies used were PE-Cy7 conjugated
anti-IFN-.gamma. (clone B27, BD biosciences), Pacific blue
conjugated anti IL17A (clone BL168, Biolegend), Alexa fluor 700
anti IL-2 (clone MQ1-17H12, Biolegend), PE conjugated anti IL-4
(clone 8D4-8, BD Biosciences), AF 488 conjugated TNF-.alpha.,
anti-CD3 Qdot 605 (clone UCHT1, Invitrogen) and PE-Cy5 anti-CD69
(clone FN50, BD biosciences). After intracellular staining, cells
were further washed 3-times with 1.times. permeabilization buffer
and one final wash with FACS buffer before resuspending them into
the FACS tubes. A custom made BD LSR II flow cytometer equipped for
the detection of 12 fluorescent parameters was used to collect
2-5.times.10.sup.5 events for each sample and data was analyzed
using FLOW JO (Tree Star) software. Gates for cytokine positive
cells were determined by the help of unstimulated and SEB
stimulated cells and cytokine responders were confirmed by
excessive back-gating.
v. Humoral Responses
[0286] For measuring IgG antibody levels in the samples, ELISA was
performed as described previously [Pichichero et al., Vaccine 2010;
28:7184-7192]. Briefly, 96-well ELISA plates (Nunc-Immulon) were
coated with 0.5 .mu.g/ml of individual antigens (100 .mu.l/well) in
coating buffer (bicarbonate, [pH 9.4]) and incubated overnight at
4.degree. C. After washing, the plates were blocked with 3% skimmed
milk at 37.degree. C. for 1 hr (200 .mu.l per well). After five
washes, 100 .mu.l of serum at a starting dilution of 1:100 (in
PBS-3% skim milk) was added to the wells and diluted serially 2
fold. The mixture was incubated at room temperature for 1 hr
followed by the addition of affinity purified goat anti-human IgG,
IgM or IgA antibody conjugated to horseradish-peroxidase (Bethyl
Laboratories, Inc, Montgomery, Tex.) as a secondary antibody. The
reaction products were developed with TMB Microwell Peroxidase
Substrate System (KPL, Gaithersburg, Md.), stopped by the addition
of 1.0 molar phosphoric acid and read by an automated ELISA reader
using a 450-nm filter. To provide quantitative results on antibody
concentrations, the level of the specific antibody present in the
unknown sample was determined by comparison to an internal
reference serum (pool of human serum with high antigen titers). The
levels of IgG in the reference serum were quantitatively measured
by using a human IgG ELISA quantitation kit (Bethyl laboratories).
A Four-parameter logistic-log function was used to form the
reference and sample curves.
vi. Statistics
[0287] All data was analyzed using Graph Pad Prism software. Two
tailed P values for the data were calculated using Mann Whitney
Test. Percentages of atopy and non-atopy between all non-otitis
prone and otitis prone children were compared using chi-square
method.
3. Results
[0288] i. Study Population
[0289] From a total study population of 387 children 19 otitis
prone children were identified. From the remainder children those
with 1 or 2 AOMs who were of a similar age as the non-otitis prone
children were randomly selected as comparators for the study.
Clinical characteristics of the study children are shown in Table
2. No significant differences were found in atopic and non atopic
children between the two cohorts (p=0.5).
TABLE-US-00005 TABLE 2 Basic characteristics of Study subjects
Pneumococcal NTHi Otitis Non-otitis Otitis Non-otitis prone prone
prone prone (n = 13) (n = 14) (n = 6) (n = 6) Gender Male 9 10 3 2
Female 4 4 3 4 Mean Age 13.5 10.1 14.3 8.2 (months) Number of AOM
episodes >3 in 6 months 6 0 5 0 >4 in 12 months 7 0 1 0
Number of NP colonizations with respective pathogen 1-3 10 10 4 5
4-5 3 1 1 0 6 or more 0 0 1 0 Ventilation tube 4 None 3 None
placement Adenoidectomy None None None None Breast feeding 12 8 3 1
Atopy 7 4 4 1 Non Atopic 6 10 2 5
ii. Otitis Prone Children have Reduced Percentages of Antigen
Specific Functional T-Helper Memory Responses to Spn and NTHi in
their Circulation.
[0290] The circulating frequencies of various Spn and NTHi
antigen-specific memory T-helper cell subsets were compared between
non-otitis prone and otitis prone children by stimulating their
PBMCs with specific antigens. For that, the percentages of T-helper
memory cells producing IFN-.gamma., IL-4, IL-2 or IL-17 were
calculated by gating on activated CD69.sup.+ T-cells (FIG. 15). No
difference was found in the naive and memory CD4.sup.+ T-cell
counts among both the cohorts (Table 3). Antigen specific responses
were normalized with the control PBMCs left unstimulated or
stimulated with a non-specific antigen (Keyhole limpet
hemocyanin).
TABLE-US-00006 TABLE 3 Circulating CD4.sup.+ T-cell counts per
million PBMCs in otitis prone and non-otitis prone children Cell
counts per microliter (.mu.1) of blood (Mean .+-. SD) Non-otitis
Otitis prone prone P Cell type (n = 19) (n = 20) values
CD3.sup.+CD4.sup.+ T-cells 2306 .+-. 452 2099 .+-. 380 0.17
CD3.sup.+CD4.sup.+CD45RA.sup.+ 1322 .+-. 640 1240 .+-. 590 0.10
(naive CD4.sup.+ T-cells) CD3.sup.+CD4.sup.+CD45RA.sup.- 455 .+-.
180 537 .+-. 198 0.18 (memory CD4.sup.+ T-cells)
[0291] FIG. 16A demonstrates frequencies of the various subsets of
T-helper memory cells to all the Spn antigens used for stimulation
in non-otitis prone children (n=15) following AOM (n=6) or NP
colonization (n=9) with Spn. In sharp contrast, otitis prone
children (n=13) had a marked dysfunction of circulating Spn
specific T-helper memory cells after AOM (n=10) and NP colonization
(n=3). In particular, there was a complete lack of T-helper memory
cells producing IFN-.gamma. against LytB, PhtE and PlyD1 whereas
significantly lower levels of IFN-.gamma. were produced in response
to PhtD, PcpA and PspA (P<0.02). A significant decrease in IL-4
producing T-helper memory cells was observed against PhtD and LytB
(P<0.02) in the otitis prone children. IL-2 responses to PhtD
(P<0.05), PcpA (P<0.005), PhtE (P<0.05), PlyD1
(P<0.005) and PspA (0.02) were significantly lower in otitis
prone children and a significant reduction in IL-17a producing
cells were found in otitis prone children in response to PhtD, PcpA
and PhtE (P<0.05).
[0292] FIGS. 3A-D show the results of a separate series of
experiments involving 6-non-otitis prone children (all NP colonized
with NTHi) and 6-otitis prone children either NP colonized with
NTHi (n=2) or having an AOM episode caused by NTHi (n=4). PBMCs
were stimulated with NTHi protein antigens P6, OPM26 and protein D.
Otitis prone children were devoid of IFN-.gamma. producing T-helper
memory cells against all 3 NTHi antigens used for stimulations.
Otitis prone children lacked an IL-4 response to P6 antigen
(p<0.05) but no significant differences were observed in the
IL-4 response to OMP26 and protein D compared to non-otitis prone
children (p=0.6). No T-helper memory cells were found in otitis
prone children producing IL-2 upon stimulation with protein D, and
the frequencies of cells responding to OMP26 and P6 were
significantly reduced (p<0.05).
[0293] Neither otitis prone nor non-otitis prone children showed
IL-17a response upon stimulation with P6. Otitis prone children
were devoid of OMP26 specific memory Th-cells producing IL-17a, a
significant difference from non-otitis prone children (p=0.05). The
difference in the frequencies of IL-17a producing memory T-helper
cells to protein D was not significant (P=0.7).
iii. Otitis Prone Children are not Deficient in Total Functional
Memory T-Cells
[0294] Impaired T-helper memory cell responses to the Spn and NTHi
antigens among otitis prone children were due to intrinsic T-cell
defects among otitis prone children. For that, PBMC were stimulated
with SEB (as described in methods), an antigen that stimulates a
T-cell response independent of antigen presenting cell involvement
[Llewelyn et al., Int. Immunol. 2006; 18(10):1433-41]. Upon
stimulation with SEB, percentage of CD45RA.sup.Low CD4.sup.+
T-cells producing IFN-.gamma., IL-4, IL-2 or IL-17a was found to be
the same for otitis prone and non-otitis prone children (P>0.5;
FIG. 16B).
iv. Antibody Responses to Spn and NTHi Protein Antigens are Reduced
in Otitis Prone Children.
[0295] Antigen specific IgG titers were evaluated in the serum of
non-otitis prone and otitis prone children. Serum IgG levels to the
similar Spn and NTHi antigens in the respective groups are shown in
FIG. 17. As expected, with the increased T-helper memory cell
frequencies, IgG titers to PhtD, LytB, PhtE, PlyD1 were
significantly higher in the non-otitis prone group compared to
otitis prone (P<0.05; 0.0005; 0.0005; 0.005 respectively),
whereas PcpA levels were not significantly different between the
groups (FIG. 17A). Among NTHi antigens significantly higher IgG
levels were observed to Protein D in non-otitis prone children
compared to the otitis prone children (p<0.05), whereas no
significant differences in the levels of IgG antibody to P6 and
OMP26 were measured between the groups (FIG. 17B).
4. Discussion
[0296] Children who experience repeated AOM suffer the greatest
morbidity from this infection, sometimes resulting in permanent
hearing loss [Vergison et al., Lancet Infect. Dis. 2010;
10(3):195-203; Morris and Leach, Pediatr. Clin. North Am. 2009;
56(6):1383-99]. As compared to non-otitis prone children, previous
reports have described otitis prone children to produce lower
amounts of Spn and NTHi-specific antibodies and/or not to produce
functional bactericidal antibodies [Faden, Eur. J. Pediatr. 2001;
160(7):407-13; Pichichero et al., Vaccine 2010; 28:7184-7192;
Murphy and Yi, Ann. N.Y. Acad. Sci. 1997; 830:353-60]. These
findings indicated that decreased concentration of circulating
antibodies to the otopathogen antigens explained the otitis prone
condition. A more precise immunological explanation for the
observed lower antibody levels in the otitis prone children to was
sought to facilitate further research to circumvent the
dysfunction. It was postulated that a reduced antibody response
observed in the otitis prone children can be the result of impaired
CD4.sup.+ T-helper cell responses to the pathogen. Hence,
generation of antigen specific memory CD4.sup.+ T helper cell
subsets (Th-1, Th-2 and Th-17) were compared between non-otitis
prone and otitis prone populations of children. This becomes
important since CD4.sup.+ T helper cells have been shown to mediate
help in fighting infections caused by Spn and NTHi [Malley et al.,
Infect. Immun. 2006; 74(4):2187-95; King et al., Am. J. Respir.
Crit Care Med. 2003; 167(4):587-92; de Bree et al., J. Infect. Dis.
2007; 195(11):1718-25]. However, there is no report demonstrating a
protective role of pathogen-specific CD4.sup.+ T helper-cells in
AOM in children which is caused by these respiratory pathogens.
[0297] A clear reduction in the functional memory CD4.sup.+ T cell
frequencies producing various cytokines among children that are
prone to AOM infections was found (FIGS. 16A and 3A-C). Otitis
prone children develop short-lived antibody responses since
antibodies were detectable among these children after AOM and NP
colonization with otopathogens (FIG. 17A-B). However, in the
absence of adequate pathogen-specific memory CD4.sup.+ T cell
frequencies and after the antibody levels wane the child quickly
becomes susceptible to additional AOM infections. Recent work on
follicular T helper cells (Tfh) has established their significance
in generating B-cell mediated antibody responses. Hence, it was
expected that otitis prone children can have reduced Tfh in their
circulation. Surprisingly, staining of CD4.sup.+ T cells for CXCR5
expression did not identify a difference in the Tfh population in
the circulation in otitis prone or non-otitis prone children (data
not shown). At first, only a low percentage of CXCR5 expressing CD4
Tfh-cells can be detected in the peripheral blood as demonstrated
in adults [Fazilleau et al., Immunity. 2009; 30(3):324-35].
Secondly, preliminary data indicate that children of this age group
lack overall CXCR5 expressing CD4.sup.+ T cells in their
circulation. This makes it difficult to compare Tfh populations in
the PBMCs of otitis prone and non-otitis prone children
(unpublished data). Furthermore, as a result of SEB stimulation,
similar percentages of functional memory CD4.sup.+ T cells were
observed among both the cohorts and that rules out an intrinsic
defect in the CD4.sup.+ T cells of otitis prone children (FIG.
1C).
[0298] Previous work has demonstrated role of Spn and NTHi antigens
in CD4.sup.+ T cell proliferative responses (for 5-7 days) among
children and adults [Mureithi et al., J. Infect. Dis. 2009;
200(5):783-93; Zhang et al., J. Infect. Dis. 2007;
195(8):1194-202]. A prior study evaluated CD4.sup.+ T cell
proliferation in the cells collected from the adenoids and tonsils
of otitis prone children and found no proliferation in response to
NTHi protein P6 [Kodama et al., Acta Otolaryngol. 1999;
119(3):377-83]. Studies of this nature are imperative to evaluate
antigen specific T cell proliferation but fail to inform about
occurrence of antigen specific memory CD4.sup.+ T-cells. These data
are the first report that demonstrates increased frequencies of Spn
and NTHi-specific IL-17a producing memory Th-cells in the
circulation of non-otitis prone children, as compared to otitis
prone children (FIG. 1). Although not directly demonstrated, the
IL-17a producing memory Th-cells can contribute to protection
against the otitis prone condition caused by Spn or NTHi by an
antibody-independent mechanism as demonstrated in a mouse model
[Malley et al., Infect. Immun. 2006; 74(4):2187-95].
[0299] The cellular phenotyping of middle ear fluid during AOM as
well as adenoids in similar individuals has indicated a large
migration of CD45RO.sup.High/CD45RA.sup.Low memory CD4.sup.+
T-cells as determined by loss of homing receptors L-selectin
[Mattila et al., Int. Immunol. 2000; 12(9):1235-43; Skotnicka et
al., Otol. Neurotol. 2005; 26(4):567-71]. Local secondary lymphoid
organs such as adenoids are the primary sites for T-cell priming
during upper respiratory tract bacterial infections and
nasopharyngeal colonization. Once, an antigen loaded APC migrates
to local lymphoid organs (adenoids), the differentiation of
lymphocytes (c.f. CD4.sup.+ T-cells) takes place. After entering
the blood circulation the CD4.sup.+ T-cells may eventually migrate
to the middle ear mucosa (in case of AOM) and/or the upper
respiratory tract (during NP colonization) [de Bree et al., J.
Infect. Dis. 2007; 195(11):1718-25; Mattila et al., Int. Immunol.
2000; 12(9):1235-43]. Unlike mice, it is practically impossible to
track antigen-specific CD4.sup.+ T-cells in human subjects.
Nevertheless, evaluation of MEF for the cellular phenotypes
indicates that T-helper memory cells may play a key role in the
elimination of AOM pathogens at the middle ear mucosa. Hence
otopathogen-specific T cell memory, if generated, can be helpful in
the prevention of recurrent AOM.
[0300] A decreased antibody response has been reported previously
after immunization with rubella vaccine in otitis prone children
[Prellner et al., Ann. Otol. Rhinol. Laryngol. 1990; 99(8):628-32].
A similar dysfunction in T cell responses to vaccination have been
observed among bone marrow or stem cell recipients [Avetisyan et
al., Bone Marrow Transplant. 2005; 36(5):411-5; Avigan et al.,
Biol. Blood Marrow Transplant. 2001; 7(3):171-83]. Also earlier
studies have suggested a genetic polymorphism in the expression of
various immunoresponsive genes TNFa, IL-6, IL-10 among otitis-prone
children [Emonts et al., Pediatrics 2007; 120(4):814-23; Revai et
al., Clin. Infect. Dis. 2009; 49(2):257-61]. Faulty function of
APCs has been described to be responsible for immature T cell
responses among infants and young children [Zaghouani et al.,
Trends Immunol. 2009; 30(12):585-91]. Furthermore, dendritic cells
in infants has been shown to pose restriction in generating
vaccine-specific T cell memory [Upham et al., Infect. Immun. 2006;
74(2):1106-12]. Collectively, based on the presented data as well
as prior reports it is possible that APCs in otitis prone children
are unable to prime naive T cells for memory generation. Whether
otitis-prone children possess an immature subset of APC and
therefore are unable to process and present antigens to the
CD4.sup.+ T-cells for effector/memory generation are now an area of
investigation.
[0301] Generating efficacious immunity against NTHi requires CD4 T
cells directed against the otopathogen. peripheral blood
mononuclear cells (PBMCs) were used to discover that OP children
have limited pathogen-(NTHi) specific memory CD4 T cells as
compared to NOP children (FIG. 3). These findings point the way to
a need for specific protein structures and adjuvants to enhance the
immune response in OP children.
[0302] This study aimed at determining the divergent generation and
function of T-helper effector/memory as a mechanism for failure to
prevent AOM in OP children. A multi-parameter ICCS flow cytometric
method was developed. Using P6 and 2 other NTHi and 6 Spn protein
antigens, NTHi and Spn-specific functional CD4.sup.+ T-helper
effector/memory cell subsets were analyzed from the peripheral
blood, adenoids and tonsils of OP and NOP children. These data show
a consistent lack or reduction of NTHi and Spn antigen specific
memory (CD45RA.sup.Low) CD4 T cells among OP children in the blood.
This indicated that the lack of functional memory CD4 T-cell
responses in the OP child makes them susceptible to repeated AOMs
until a more developed immunologic maturation is achieved (Sharma
et al JID 2011 in press). The exception is P6 where functional
antibody responses are generated from natural infection in OP
children infected with NTHi [Khan et al., FEMS Immunol Med
Microbiol 2012 DOI:10.1111/j.1574-695X.2012.00967.x].
[0303] An absence/lower proliferation and cytokine production was
found to many antigens but less so to NTHi P6 in the OP children as
compared to NOP children. In the B cells, a lower/deficiency of B
cell memory generation was found in OP children except less so to
P6 [Sharma et al., J Infect Dis. (2012) 205(8):1225-1229].
C. Example 3
AOM and Inflammation
[0304] The innate response is comprised of both innate gene
responses from epithelium and innate immune (NK, neutrophil,
macrophage) responses that release pro-inflammatory cytokines that
affect epithelial inflammation. There is a 93% correlation in
children experiencing an active AOM infection and a concurrent
respiratory viral infection. Viral infections drive significant
changes to the airway epithelium manifested by morphological
changes and inflammation induced by inflammasomes or cytokines, and
cell death. These events set the stage for AOM. In addition,
respiratory bacteria also cause inflammation and epithelial cell
death. Currently, the role that NHTi plays in driving inflammasome
activation of epithelium is not known but colonization is
associated with increases in epithelial inflammation. These data
show a trend in higher NLRP3 activation after NTHi infection in OP
children (FIG. 4). The alterations to nasal epithelium to permit
the transition from colonization to pathogenic infection have not
been determined although viral infections of nasal epithelial cell
lines cause up-regulation of epithelial cell receptors for
bacterial adherence to occur more effectively. FIG. 5 shows PAFr, a
bacterial adherence factor appears more up-regulated (trend) in OP
than NOP children during AOM infection.
[0305] The exact mechanisms facilitating the transition from
otopathogen colonization to AOM has not been fully determined
although there is an association between the levels or
polymorphisms of innate cytokine expression and AOM in OP children.
The onset of AOM in OP and NOP children generally occurs within the
first 6 days of a viral infection when there is significant innate
cellular recruitment to the respiratory mucosa which is associated
with strong pro-inflammatory cytokine release. The levels of these
pro-inflammatory cytokines to viral infections could dramatically
favor bacterial colonization by stressing nasal epithelium and
driving progression to AOM. OP children can have divergent
proinflammatory cytokine responses (FIG. 6) which may be due to
either a difference in innate cytokine responses or by a lack of
T-cell memory (FIG. 3) which would fail to rapidly reduce antigen
burdens as would occur in NOP children and drive continued
heightened epithelial stress.
D. Example 4
Toll-Like Receptors and AOM
[0306] Toll-like receptor (TLR) expression plays a role in
initiating the activation of the immune response to bacterial
infection. Previous studies have determined a correlation between
disruption of TLR4 or MyD88 and AOM (Hernandez et al., (2008). J
Infect Dis. 198 (12):1862-9). Differences in TLR expression have
also been demonstrated to be a component of immunological
maturation and adequate immune stimulation during infection.
Therefore, children prone to AOM can have recurrent AOM due to
intrinsic TLR expression deficiency. Defective innate immunity is
associated with poor sepsis outcome in neonates but is reversible
with TLR agonists implying that the capacity to respond to TLR
stimulation may be a critical factor in OP children with immune
competency that potentially mirrors neonates. FIG. 7 shows
different TLR expression levels in cells recruited to the nasal
mucosa during AOM in OP and NOP children. Surprisingly, it was
determined that OP children have higher expression levels of TLR2
and TLR4 despite evidence that APC function may be lower which
could imply downstream defects in signaling.
[0307] These data identify targets to be used as therapeutics with
P6 to further enhance immunogenicity, and to overcome differences
in immune responses of OP children in the levels of
pro-inflammatory cytokines released during the innate immune
response that drives AOM. The targets are downregulation of NLRP3,
PAFr and IL1B and for upregulation are IL6, CCL3, CCL4 and
downstream signaling of TLR2 and TLR4.
[0308] OP children with AOM infection have different levels of
inflammatory gene expression in response to viral and bacterial
co-infection than NOP children, but less so with P6 (FIG. 8).
Inflammatory conditions can disrupt epithelial membrane integrity,
lead to epithelial cell receptor alterations, and promote
epithelial cell death, events that are associated with enhanced
NTHi binding to epithelial mucosa and thereby facilitate an
increase in the otopathogen inoculum. However, lower inflammatory
conditions can also have an impact on the activation of the
adaptive immune response that is more closely associated with
resolution of infections and appears to deviate in OP and NOP
children based on our data, but less so with P6.
E. Example 5
Antibody Response to Haemophilus influenzae Outer Membrane Protein
D, P6, and OMP 26 after Nasopharyngeal Colonization and Acute
Otitis Media in Children
1. Introduction
[0309] Nontypeable Haemophilus influenzae (NTHi) is currently the
most frequent cause of episodic and recurrent acute otitis media
(AOM) in children in the United States [Casey and Pichichero,
Pediatr Infect Dis J 2004; 23(9):824-8; Pichichero and Casey,
Pediatr Infect Dis J 2007; 26(10):512-6; Casey et al., Pediatr
Infect Dis J 2010; 29(April (4)):304.9]. AOM and all respiratory
bacterial infections begin pathogenesis with colonization of the
nasopharynx (NP). However, carriage of NTHi is mostly asymptomatic;
only when the condition of the host is altered, NTHi may invade the
middle ear, causing AOM.
[0310] A vaccine against NTHi presents a different set of
challenges compared with Hib vaccination because rather than a
single dominant capsular antigen, NTHi strains express multiple
outer membrane proteins (OMPs) [Barenkamp et al., Infect Immun
1982; 36:535-40; Loeb and Smith, Infect Immun 1980; 30:709-17;
Murphy et al., J Infect Dis 1983; 147:838-46; St. Geme, Vaccine
2001; 19(1):541-50] Several of the OMPs of NTHi have been
eliminated as vaccine candidates due to surface epitope
heterogeneity, variable expression or other characteristics [St.
Geme, Vaccine 2001; 19(1):541-50; Kyd and Cripps, J Biotechnol
1999; 73:103-8]. Desirable vaccine candidate antigens for NTHi
should be conserved among strains and immunogenic in children and
adults. At this time one OMP of NTHi, protein D, has been
incorporated into a commercialized vaccine product as a carrier of
pneumococcal polysaccharide antigens. Administration of that
conjugate vaccine resulted in a 35% reduction in AOM caused by NTHi
[Prymula et al., Lancet 2006; 367:740-8]. Further proof of the
efficacy of protein D as a vaccine ingredient for prevention
of NTHi mucosal infections is needed and the study of other NTHi
antigens is underway in many laboratories, anticipating the need
for amulti-component vaccine to optimize protection at rates higher
than protein D alone.
[0311] Two additional NTHi OMPs that are leading vaccine candidates
are protein 6 (P6) and protein OMP26, since they possess the
desirable features noted above [St. Geme, Vaccine 2001;
19(1):541-50; Kyd and Cripps, J Biotechnol 1999; 73:103-8]. For
NTHi vaccine development it is important to know whether antibodies
develop after natural NTHi exposure such as after asymptomatic NP
colonization and after AOM. In the present study it was
hypothesized that NTHi NP colonization and AOM would represent
immunizing events for potential OMP vaccine ingredients. This is
the first study to prospectively compare the development of natural
antibodies to 3 NTHi outer membrane proteins D, P6 and OMP26
simultaneously in a cohort of children 6-30 months of age during NP
colonization and AOM. The comparisons of interest we report here
include: 1. Changes in the levels of protein D, P6 and
OMP26-specific IgG antibodies in children as they increased from 6
to 30 months of age; 2. Changes in antibody levels following
detected colonization of the NP with NTHi; 3. Differences in
antibody levels in convalescence from NP colonization versus AOM;
4. Variations in individual antibody repertoire and responses in
the study cohort following AOM; and 5. Differences in contribution
of antibodies to protein D, P6 and OMP26 to bactericidal
activity.
2. Methods
[0312] i. i. General design
[0313] This report includes data for the 3 year time span June,
2006 to December, 2009 from children enrolled in a 5 year
prospective study supported by the National Institutes of Deafness
and Communication Disorders. Healthy children without previous
episodes of AOM were enrolled from a middle class, suburban
socio-demographic pediatric practice in Rochester, N.Y. (Legacy
Pediatrics). Healthy children had serum, NP and oropharyngeal (OP)
cultures and NP wash samples obtained seven times, every 3.6
months, between 6 and 30 months of age (at age 6, 9, 12, 15, 18,
24, and 30 months). In addition, if a child developed symptoms
compatible with AOM, they were examined by validated otoscopist
pediatricians with pneumatic otoscopy and if middle ear infection
was suspected a tympanocentesis was performed to confirm the
diagnosis. At the time of the acute AOM diagnosis and three weeks
later acute and convalescent serum, NP and OP cultures and NP wash
samples were obtained. The study was approved by the University of
Rochester and Rochester General Hospital Research Subjects Review
Board and written informed consent was obtained for participation
and all procedures.
[0314] Three NTHi OMPs were elected to be studied. Protein D is a
highly conserved antigen among NTHi strains [Forsgren et al., Clin
Infect Dis 2008; 46:726-31]. It is a 43 kDa surface exposed
lipoprotein that has glycerophosphodiesterase. P6 has been
described as a highly conserved OMP among NTHi strains.
Immunization with P6 provides protection against AOM in the
chinchilla model [DeMaria et al., Infect Immun 1996; 64:5187-92].
OMP26 is a highly conserved protein of NTHi that is associated with
protection against NTHi infections after parenteral and mucosal
immunization in the chinchilla and rat models that induced high
levels of antibody [Kyd and Cripps, Infect Immun 1998; 66:2272-8;
Kyd et al., Infect Immun 2003; 71:4691-9].
ii. Definition of AOM
[0315] AOM was diagnosed by pneumatic otoscopy by validated
otoscopists, when children with acute onset of otalgia had tympanic
membranes (TMs) that were: (1) bulging or full; and (2) a cloudy or
purulent effusion was observed, or the TM was completely opacified;
and (3) TM mobility was reduced or absent.
iii. Tympanocentesis
[0316] MEF for culture was obtained by puncture of the inferior
portion of an intact TM with a 20-gauge spinal needle attached to a
3-mL syringe using a hand-held operating otoscope. If a small
sample of MEF was obtained on aspiration, 0.5 mL of trypticase soy
broth was aspirated through the spinal needle and then aliquoted
and inoculated onto agar plates and into broth, as described
below.
iv. Sample Collection
[0317] At each sampling visit a cotton-tipped wire swab was
inserted into both nares and a culture of the posterior nasopharynx
was obtained; an OP culture was obtained by rubbing both tonsils
and the posterior pharynx. Then 1 mL of sterile phosphate buffered
saline was instilled and aspirated from both nares as a third
sample for culture. Serum was obtained by venipuncture after
application
of ELMA cream for local numbing of the area. v. Microbiology
[0318] MEF, NP, and OP samples were inoculated into trypticase soy
broth, trypticase soy agar with 5% sheep blood plates, and
chocolate agar plates. All samples were incubated at 37.degree. C.
with 5% carbon dioxide. Bacteria were isolated according to the
CLSI standard culture procedures. An isolate was further identified
as NTHi on a similar basis as described by Murphy et al. [Murphy et
al., J Infect Dis 2007; 195:81-9] to include not only colony
morphology, porphyrin reactivity, and growth requirement for hemin
and nicotinamide adenine dinucleotide and Haemophilus ID Quad
plates, but also by ompP6 sequencing to distinguish NTHi from H.
haemolyticus [Murphy et al., J Infect Dis 2007; 195:81-9].
vi. Detection of OMP-Specific Antibodies by ELISA
[0319] Protein D, P6 and OMP26-specific antibody titers were
determined by ELISA using purified recombinant protein D (provided
as a gift from GlaxoSmithKline Biologicals, Rixensart Belgium),
lipidated P6 (provided as a gift by Dr. Tim Murphy, University of
Buffalo) and OMP26 (provided as a gift by Jennelle Kyd, University
of Can berra, Australia). 96-well Nunc-Immulon 4 plates were coated
with 0.25-0.5 .mu.g/mL of individual OMP antigens 100 .mu.L/well)
in coating buffer (bicarbonate, [pH 9.4]) and incubated overnight
at 4.degree. C. After washing the plates were blocked with 3% skim
milk at 37.degree. C. for 1 h (200 .mu.L per well). After five
washes, 100 .mu.L of serum at a starting dilution of 1:100 (in PBS.
3% skim milk) was added to the wells and diluted serially 2 fold.
The mixture was incubated at room temperature for 1 h followed by
the addition of affinity purified goat anti-human IgG, IgM or IgA
antibody conjugated to hoarseradish-peroxidase (Bethyl
Laboratories, Inc., Montgomery, Tex.) as a secondary antibody. The
reaction products were developed with TMB Microwell Peroxidase
Substrate System (KPL, Gaithersburg, Md.), stopped by the addition
of 1.0M phosphoric acid and read by an automated ELISA reader using
a 450-nm filter.
[0320] To provide quantitative results on antibody concentrations,
the level of the specific antibody present in the unknown sample
was determined by comparison to an internal reference serum (pool
of human serum with high anti-OMP titers). The levels of IgG, IgM
and IgA in the reference serum were quantitatively measured by
using a human IgG/IgA/IgM ELISA quantitation kit (Bethyl
laboratories).
[0321] A four-parameter logistic-log function was used to form the
reference and sample curves. This ELISA was fully validated
according to ICH Guidance. The assay lower limit of detection for
protein D was 3.5 ng/mL for IgG, 4.5 ng/mL for IgM, and 8 ng/mL for
IgA; for P6 it was at 1 ng/mL for IgG, 3 ng/mL for IgM, and 3 ng/mL
for IgA; and for OMP26 it was at 4 ng/mL for IgG, 3 ng/mL for IgM,
and 10.5 ng/mL for IgA. The inter-assay coefficient of variation
was 20% for all antigens and secondary antibody combinations.
vii. Bactericidal Assay
[0322] Eleven sera were randomly selected from those with the
greatest volumes to measure bactericidal activity pre and post
absorption with protein D, P6 and OMP26. The sera were
heatinactivated at 56.degree. C. for 30 min to eliminate human
complement. Each serum was assayed against the bacterial strain
isolated from middle ear space of that child. Homologous NTHi
strains were cultivated, harvested, and diluted to a concentration
of .about.10.sup.5 CFU/mL. Twelve serial twofold dilutions of the
serum to be tested (starting at 1:2) were mixed with precolostral
calf serum complement and 20 .mu.L of bacteria. After 60 min of
incubation, the number of surviving bacteria was determined by
plating 5 .mu.L onto chocolate agar and counting the colonies. The
bactericidal titer of the serum was defined as the inverse of the
highest dilution that led to .gtoreq.50% bacterial killing and was
compared to that of negative control serum. Appropriate controls
were included in all experiments. To examine the contribution of
protein D, P6 and OMP26 antibodies to serum bactericidal activity
observed, we removed all protein D, P6 or OMP26 antibodies from
available sera using polystyrene beads. For the absorption
procedure, polystyrene beads were washed extensively with borate
buffer (pH 8.5) and resuspended in 1 mL of Borate buffer. Freshly
prepared recombinant protein D, P6 and OMP26 antigens were
incubated with these beads overnight at room temperature. The beads
were washed extensively, incubated in BSA/Borate buffer for 30 min
at room temperature, then pelleted and incubated with 200 .mu.L of
patient sera for 2 h at room temperature. The beads were
centrifuged (200.times.g) and the supernatant was collected.
[0323] The efficacy of absorption was monitored by using ELISA as
described above. The reciprocal bactericidal titers were compared
with unabsorbed sera to determine the bactericidal activity
mediated by each of the specific antibodies. An experiment was
performed with cross adsorption of other antibodies and found that
adsorption was quite specific.
viii. Multi-Locus Sequence Typing (MLST)
[0324] Bacterial genomic DNA was extracted from pure cultures of
NTHi isolated from NP, OP or MEF samples (If children have AOM).
The internal fragments of seven housekeeping genes of NTHi were
amplified by PCR, using PCR Master Mix (Promega, 50 units/mL of Taq
DNA Polymerase, 400 .mu.M dNTP, 3 mM MgCl.sub.2.) using primers
described previously [Medeiros et al., Rev Inst Med Trop Sao Paulo
1998; 40:7-9]. PCR conditions were as follows: initial denaturation
at 95.degree. C. for 4 min, followed by 30 cycles of 95.degree. C.
for 30 s, 50-55.degree. C. annealing for 30 s and 72.degree. C.
extension for 30 s.
[0325] Sizes of PCR products were checked by running 1.5% agarose
gel electrophoresis stained with ethidium bromide. The size of PCR
products was 50-100 bps larger than the fragments for typing. The
PCR products were purified using the Exo-sap kit (USB Company) and
identified by DNA sequencing. Sequencing analyses were performed on
an ABI Prism 3730.times.1DNA analyzers with the same primers used
for PCR product amplification.
ix. Statistics
[0326] Two sample comparisons were performed using either paired
t-test, two-sample t-test (using a log transformation where
appropriate), the Mann-Whitney rank sum test or Fisher's exact
test. Testing for increasing antibody level with age (FIG. 10) was
complicated by inconsistently represented time points, and by data
dependence induced by repeated sampling by subject. A modification
of Kendall's tau statistic was used to measure within-subject
concordance of level with age. Any pair of within subject antibody
level measurements which increases with age was concordant, and was
discordant otherwise (neither applies in the case of ties). In the
calculation Nc and Nd were the number of concordant and discordant
within-subject pairs and G=(Nc-Nd)/(Nc+Nd). The statistic G was
calibrated as a correlation, with -1 and 1 representing perfect
negative and positive associations, respectively, between age and
antibody level measurement. The quantity S=Nc/(Nc+Nd) represented
the proportion of increasing pairs. If there was no concordance
S=1/2 was expected, on average. To assess significance, a bootstrap
procedure was performed by resampling subjects with replacement.
This procedure permitted within-subject dependence. The bootstrap
sample was then used to estimate 95% confidence intervals for S,
and p-values against the null hypothesis S=1/2.
[0327] For all testing, p<0.05 was considered significant.
3. Results
[0328] i. NP colonization and AOM events
[0329] During the 3 years of enrollment a total of 130 children
were recruited into the study. There were 631 visits with 72 NTHi
OP/NP colonization episodes documented in 47 (28%) children who
were culture-positive for NTHi at one or more of the seven sampling
visits (FIG. 9). Among the 47 NP colonized children, 31 (66%) of
the children were NTHi culture-positive at one sampling visit, 10
(21%) at two visits, 4 (9%) at three visits and 2 (4%) at four or
more visits. Eighty three (64%) did not have NTHi detected by
culture in the NP or OP at any of the 7 visits. Thirty-seven (79%)
children experienced colonization that was detected by culture and
cleared by the next sampling 3-6 months later. NP colonization was
detected at the first sampling at 6 months of age in 10 (21%)
children and at various times thereafter for the remainder of the
subjects. Nine of the 47 colonized children (19%) experienced
prolonged detected colonization of 6 months or longer with the same
NTHi strain based on multi-locus sequence typing (subjects 1, 6, 7,
19, 20, 22, 37, 40, and 44; MLST data not shown). Because the study
design called for NP/OP sampling at 7 specific times separated by
3-6 months, some NTHi colonization events were not detected by
culture but most likely occurred as reflected in significant rises
in specific antibody to one or more of the NTHi antigens studied.
There were 28 NTHi AOM episodes in 18 children. Nine (50%) of the
18 AOM children experienced one AOM due to NTHI, 8 (44%) children
experienced 2 AOM events due to NTHi and 1(6%) experienced 3 NTHi
AOM events (FIG. 9).
ii. Natural Acquisition of Serum Antibody to Protein D, P6 and OMP
26 Over Time
[0330] FIG. 10 shows a boxplot of the measured serum antibodies to
protein D, P6 and OMP26 at 6, 9, 12, 15, 18, 24, and 30 months of
age corresponding to visits 1-7 for the study cohort. For
comparison the antibody levels of 26 adults (age 18-60 years) are
also displayed. The antibody levels increase significantly over
time to all 3 NTHi proteins (p<0.001). Antibody levels among
each of the 3 proteins at each of the 7 time points did not differ
significantly. Compared to adults, antibody in the 24-30 month age
groups combined was significantly lower than adults for protein D
(p<0.001).
iii. Comparison of Serum Antibody Levels to Protein D, P6 and OMP26
in NP Colonized Versus Non-Colonized Children at Various Ages
[0331] The level of antibody to protein D, P6 and OMP26 was
compared in children who were NP culture-positive for NTHi at age
6, 9, 15, 18, and 24-30 months with children of the same age who
were not NP culture-positive. The patterns were significantly
different for the 3 proteins (FIG. 11). For protein D no difference
was identified between colonized and non-colonized children at age
6 or 9 months, but a significant difference was shown at age 15,
18, and 24-30 months. For P6, a significant difference was
identified between colonized and non-colonized children at age 6, 9
and 15 months, but the difference was no longer significant at age
18, and 24-30 months, largely because of wide variation in antibody
quantity among those children. For OMP26, no difference was seen
between colonized and non-colonized children.
iv. Comparison of Convalescent Serum IgG Levels to NTHi Outer
Membrane Proteins D, P6 and OMP26 Following an AOM or an NP
Colonization Event
[0332] AOM events stimulate a high quantity of antibody to the
studied proteins in convalescence. It is known that NP colonization
is a necessary first step in pathogenesis for AOM. Therefore NP
colonization plus an acute infection in the form of AOM can result
in higher antibody levels than NP colonization only. Unexpectedly
the levels of antibody to protein D, P6 and OMP26 following an AOM
episode were determined to be generally low compared to those
observed following NP colonization (FIG. 12). The lower AOM
convalescent antibody levels compared to colonization convalescent
antibody levels was significant for P6 (p<0.001) but not for
protein D or OMP26. The analysis is complicated by the fact that
the post AOM serum antibody levels were all obtained 3 weeks after
an AOM event whereas the convalescent colonization antibody levels
were obtained at the next scheduled visit for the child. Therefore
a post colonization sample could have been obtained 3-6 months
after the colonization event, at a time when some diminution of
antibody likely occurred compared to the 3 weeks post AOM sampling.
The differences in timing contributed to the variation in
convalescent antibody levels after colonization.
v. Paired Acute and Convalescent Serum IgG, IgM and IgA Antibody
Levels to NTHi Outer Membrane Proteins D, P6 and OMP26 in Children
with AOM
[0333] The quantity of total immunoglobulin to protein D
(IgG+IgM+IgA) in acute sera at the time a child presented for
clinical care for AOM was 2082 ng/mL, for P6 it was 1422 ng/mL, and
for OMP26 it was 1545 ng/mL, p=0.18 for protein D vs. P6 (FIG. 13).
In the convalescent serum, the quantity of total immunoglobulin to
protein D was 2872 ng/mL, for P6 it was 1108 ng/mL, and for OMP26
it was 2264 ng/mL, p=0.04 for protein D vs. P6. The ratio of the
three immunoglobulin classes (IgG to IgM to IgA) was not different
for the 3 proteins. When assessed as a cohort the increases in IgG,
IgM and IgA in acute vs. convalescent sera were not significant for
any of the 3 proteins.
[0334] From the data displayed in FIG. 12 for all three proteins it
appears that in the convalescent phase the antibody levels
following AOM infection are not as high as after NP colonization
and from FIG. 13 the acute to convalescent change in antibody
concentration is not significant for the overall cohort. Therefore
FIG. 5 shows the individual responses of children to all three
proteins. When the individual responses were examined where paired
sera were available it was observed that about one-third of
children showed a rising antibody response to one or more of the
NTHi antigens, with IgM predominating in acute sera (indicating a
primary immune response), one-third had no change in antibody level
between acute and convalescent sera, with IgG>IgM but relatively
high IgM (indicating that a primary response was occurring but the
sampling missed the true onset of the immune response) and
one-third had a falling antibody level, with IgG IgM (indicating
that the samples were taken well past the onset of the primary
immune response or that a secondary response had occurred). The
absence of synchrony of the antibody responses to protein D, P6 and
OMP26 is notable but at this time there are too few children to
effectively analyze this effect (FIG. 14).
vi. Bactericidal Activity of NTHi Anti Protein D, P6 and OMP26
Antibodies
[0335] To evaluate the functionality of the antibodies detected by
ELISA, bactericidal activity of the antibodies was assessed,
specific to each protein. In Table 4a the concentration of protein
D specific antibody determined by the ELISA and the bactericidal
activity of the total antibody to the homologous infecting AOM
strain is shown and compared to the concentrations and bactericidal
titers after absorption of all or nearly all of the protein D
specific antibody. For 7 of the 11 sera tested absorption of
anti-protein D antibody resulted in a significant drop in
bactericidal antibody. Table 4b shows the results for P6 (9 of 11
sera showed a drop) and Table 4c shows the results for OMP26 (0 of
11 showed a drop). The number of sera with bactericidal antibody to
protein D was significantly greater than OMP26 (p=0.01) and the
bactericidal antibody to P6 was significantly greater than OMP26
(p=0.001). Antibody to protein D and P6 accounted for all of the
detected bactericidal antibody in 5 (45%) of 11 studied children
and at least 50% of the bactericidal antibody in the remaining 6
children. To study the specificity of the cross absorption, P6 and
OMP26 ELISA titers were quantified in sera that were absorbed to
remove protein D antibodies; there was no cross absorption. Similar
experiments in duplicate were done with each serum on two different
occasions to verify the specificity of absorption for the
antibodies studied.
TABLE-US-00007 TABLE 4a Bactericidal activity of NTHi anti protein
D antibodies Anti PROTEIN Bactericidal Anti D ELISA titer* PROTEIN
Bactericical Ab titers (post (with D ELISA titer* protein D-Ab
protein D Sub- Ab titers (pre adsorption) adsorbed jects (EU/mL)
adsorption) (EU/mL) sera 1 1100 8 200 4 2 76 8 40 4 3 1500 16 100 8
4 780 16 187 4 5 773 8 291 8 6 1130 32 <5 32 7 1260 4 <5 4 8
1275 64 150 32 9 5450 16 <5 8 10 8326 8 495 8 11 213 8 <5
4
TABLE-US-00008 TABLE 4b Bactericidal activity of NTHi anti protein
P6 antibodies P6 ELISA Bactericidal Anti P6 ELISA Bactericidal Ab
titers titer* Ab titers in sera titer* (with P6 Sub- in sera (pre
(post P6-Ab adsorbed jects (EU/mL) adsorption adsorption) (EU/mL)
sera) 1 4531 8 124 4 2 180 8 91 4 3 199 16 127 8 4 1265 16 226 16 5
1160 8 192 4 6 632 32 174 8 7 933 4 38 0 8 1348 64 128 16 9
>6000 16 40 16 10 1605 8 62 4 11 674 8 32 0
TABLE-US-00009 TABLE 4c Bactericidal activity of NTHi anti OMP26
antibodies Anti Bactericidal Anti OMP26 Ab Bactericidal OMP26
titer* titers (post OMP26 titer Sub- Ab titers (pre Ab adsorption)
(with OMP26 jects (EU/mL) adsorption) (EU/mL) adsorbed sera) 1 260
8 <5 8 2 146 8 <5 8 3 281 16 <5 16 4 1110 16 265 16 5 207
8 <5 8 6 267 32 <5 32 7 1600 4 866 4 8 840 64 <5 64 9 1800
16 172 16 10 1550 8 195 8 11 243 8 <5 8 *Bactericidal titer
values are expected as a reciprocal liter of the dilution where 50%
bacterial killing was achieved.
4. Discussion
[0336] The systemic antibody response in children who experience
NTHi colonization and AOM has not been well characterized. Progress
in the development of an NTHi vaccine to prevent AOM is hampered by
our gaps in knowledge of the immune response mounted by children
who experience NTHi NP colonization and AOM. Studies in the past
often did not have the advantage of current microbiology, molecular
biology and immunology techniques, the antibody repertoire and
functionality of antibody was not fully assessed, and/or the
diagnostic accuracy of AOM and differentiation of AOM from OME--a
distinctly different clinical condition, was often not made [Shurin
et al., J Pediatr 1980; 97(3):364-9; Novotny et al., Infect Immun
2000; 68(4):2119-28; Novotny et al., Vaccine 2002;
20(29-30):3590-7; Murphy T et al., J Clin Invest 1986;
78(4):1020-7; Harabuchi et al., J Infect Dis 1994; 170(4):862-6;
Spinola et al., J Infect Dis 1986; 154(1):100-9; Faden et al., J
Infect Dis 1995; 172(1):132-5; Sloyer et al., J Infect Dis 1975;
132(6):685-8; Faden et al., J Infect Dis 1989; 160(6):999-1004;
Bernstein et al., Otolaryngol Head Neck Surg 1997; 116(3):363-71;
Harabuchi et al., Acta Otolaryngol 1998; 118(6):826-32; Yamanaka
and Faden, J Pediatr 1993; 122(2):212-8; Hotomi et al., Acta
Otolaryngol 1999; 119(6):703-7; Sloyer et al., J Clin Microbiol
1976; 4(3):306-8; Faden et al., Infect Immun 1989; 57(11):3555-9;
Yamanaka and Faden, Acta Otolaryngol 1993; 113(4):524-9; Bernstein
et al., Otolaryngol Head Neck Surg 1991; 105(3):406-10].
[0337] NP colonization by otopathogens among children has been
studied in a study design similar to ours in the past. Faden et al.
[Faden et al., J Infect Dis 1995; 172:132-5] prospectively
evaluated NP colonization by NTHi in a cohort of 200 children from
birth to two years of age living in suburban Buffalo N.Y. NP
colonization was detected in 44% of the children, with more
frequent colonization detected in the first year of life compared
to the second year of life. Frequent acquisition of NTHi strains
with frequent clearing was observed in that study, similar to our
findings. Faden et al. included children from birth to 6 months of
age when colonization occurred relatively frequently and his group
sampled the NP more often than we did in the first year of life.
This may account for our detection rate of 28% of evaluated
children compared to their study. Indeed, we did observe
significant increases in serum antibody to one or more of the NTHi
antigens we studied occurring between study visits, suggesting that
NP colonization events occurred without detection due to NP
sampling frequency.
[0338] Importantly, the gradual acquisition of serum antibody in
children over time to vaccine candidate antigens protein D, P6 and
OMP26 demonstrates that the 3 proteins are immunogenic in infants
6-30 months of age. Such an observation is strongly supportive of
the potential of these antigens to be useful in a vaccine against
NTHi infection in children. Previously, Akkoyunia et al. [Akkoyunia
et al., Infect Immun 1996; 64:4586-92] evaluated naturally
occurring protein D antibodies and found they were low in children
below one year of age but rose between age 1 and 5 years. Yamanaka
and Faden [Yamanaka and Faden, J Pediatr 1993; 122:212-8]
prospectively studied the serum antibody levels to P6 in eight
children at ages birth, 6 months, 1, 2, 4, 6, and 10 years old and
eight adults. They found that levels increased over time and the
difference became significant when 6 month olds were compared to
four year olds.
[0339] It was previously reported that NP colonization appeared to
be an immunizing event in children relative to P6 protein [Sabirov
et al., Pediatr Res 2009; 66(5):565-70]. The significant difference
in antibody level among NP colonized compared to uncolonized
children beginning at age 15 months for protein D and 6 months for
P6 provides further evidence that NP colonization with NTHi is
associated with stimulation of serum antibody to OMPs expressed by
this bacteria. The absence of a significant increase in antibody to
OMP26 in NP colonized compared to uncolonized children was
unexpected since a gradual and significant rise in antibody to
OMP26 was observed to occur as children increased in age from 6 to
30 months old. This observation will require further study. Our
findings are in contrast to those by Spinola et al. [Spinola et
al., J Infect Dis 1986; 154(1):100-9], who prospectively followed 3
children who attended a single day care center from infancy until
early childhood obtaining NP cultures periodically. They noted that
NP colonization in children with NTHi was a dynamic process with
loss and acquisition of different strains occurring over time.
Serum IgG directed to the OMPs of NTHi did not appear to change
greatly over time, or to be correlated with NP colonization. In a
study evaluating pneumococcal NP colonization and AOM, Virolainen
et al. [Virolainen et al., Pediatr Infect Dis J 1996; 15:128-33]
evaluated serum antibodies in children with AOM to pneumolysin, a
pneumococcal protein that is a vaccine candidate. Eight of 10
children experienced a seroconversion in pneumolysin antibody
levels following AOM due to pneumococci at a median age of 20
months old. Similarly, Rapola et al. [Rapola et al., Pediatr Infect
Dis J 2001; 20:482-7] studied the serum antibody response to
pneumolysin and pneumococcal surface adhesion A (PsaA) in children
with AOM age 2 months to 2 years. Antibody levels were compared
among three groups: pneumococcal AOM, pneumococcal NP colonized and
neither NP colonized nor AOM due to pneumococci. At the time of the
sampling, children with NP colonization had the highest anti-PsaA
antibody levels, children with a current AOM were next highest,
children with no current but a past history of pneumococcal NP
colonization or AOM were third highest, and lowest were those with
no current or previous documented history of pneumococcal
colonization or AOM. Wide variations in antibody levels were
measured. The findings were similar with pneumolysin. Our study of
NTHi antibody responses to protein D, P6 and OMP26 are in agreement
with the studies of pneumococcal vaccine protein candidates. Age of
the child and preexisting antibody levels are important covariates
in predicting an antibody response to NP colonization. This may
prove true also for vaccination.
[0340] The isotypes of antibody in acute and convalescent sera
surrounding an AOM for the subset of children where there was
paired serum, IgG antibody predominated although IgM antibody
levels were also elevated. This repertoire of antibody is most
consistent with prior priming of the immune response before the
AOM; otherwise we would have expected IgM to predominate in acute
sera and a switch in Ig class to occur in convalescent sera to IgG
predominant. Analysis of individual child data allowed us to
observe that the cohort analysis masked differences in the antibody
repertoire. The addition of more children to our study in the
future may allow a clearer understanding of the proportion of
children with various antibody response characteristics. In earlier
work, Samukawa et al. [Samukawa et al., Infect Immun 2000;
68:1569-73] studied the immune response to S pneumoniae surface
proteinA (PspA) and M. catarrhalis OMP UspA in the sera of various
age groups in the general population. In the first 2 years of life
they found comparable amounts of IgG and IgM serum antibodies to
both PspA and UspA whereas in adults IgG predominated. In contrast,
when Virolainen et al. [Virolainen et al., Pediatr Infect Dis J
1996; 15:128-33] evaluated serum antibodies in children with AOM to
pneumolysin they found eight of 10 children experienced a
seroconversion in pneumolysin antibody levels, all of the IgA class
only.
[0341] Not all antibody elicited by natural exposure to NTHi may be
functional; therefore the study of the contribution of antibody to
protein D, P6 and OMP26 to bactericidal activity was of interest.
Serum bactericidal antibody is associated with protection from AOM
caused by NTHi [Shurin et al., J Pediatr 1980; 97(3):364-9; Faden
et al., J Infect Dis 1999; 160:999-1004]. Previous work has shown
that serum bactericidal antibody to the homologous strain persists
after AOM and can protect against recurrent NTHi AOM infection by
the homologous strain but cross-protection for other (heterologous)
strains generally is not induced [Shurin et al., J Pediatr 1980;
97(3):364-9; Faden et al., J Infect Dis 1999; 160:999-1004].
Forsgren has previously shown that antibody to protein D can be
bactericidal [Forsgren et al., Clin Infect Dis 2008; 46:726-31].
Murphy et al. [Murphy et al., J Clin Invest 1986; 78(4):1020-7]
assessed the role of P6 as a target of bactericidal antibody and
showed that in a pool of 6 adult sera depletion of P6 antibodies
resulted in a reduction bactericidal activity. The absence of a
bactericidal effect by OMP26 has been noted [Cripps and Otczyk, Exp
Rev Vaccines 2006; 5:517-34]. Therefore, the results are consistent
with previous reports.
[0342] A significant correlation was not found between bactericidal
titers and ELISA antibody concentrations for P6, protein D and
OMP26 antibodies. Higher ELISA antibody titers did not consistently
result in higher bactericidal titers. Particularly for OMP26 high
ELISA antibody titers did not correlate with high bactericidal
titers and the depletion of OMP26 specific antibodies did not
change bactericidal titers. The disparity in antibody quantity
measured by ELISA and bactericidal titers most likely is a
reflection of the fact that ELISA measures antibody of low and high
avidity whereas bactericidal titers largely reflect high avidity
antibody [Pollard and Levin, Lancet 2000; 356(9247):2065-6;
Maslanka et al., Infect Immun 1998; 66(6):2453-9]. Also it can be
that not all OMPs expressed by NTHi elicit bactericidal
antibodies.
[0343] The difficulty of obtaining blood from young children
repetitively between 6 and 30 months of age is considerable,
particularly when coupled with additional blood sampling for acute
and convalescent levels surrounding an AOM. Therefore, despite best
efforts, blood samples were not obtained from every child at every
visit as designed. This created windows of missing data that were
addressed statistically as possible, but the addition of more
samples from more children is ongoing and may allow further light
on some of the issues addressed in this report. The need to
evaluate mucosal immune responses and cellular responses to NTHi NP
colonization and AOM was recognized and to compare immune responses
between children with absent or infrequent AOM with otitis prone
children. Those studies are ongoing and will be reported
subsequently. The study cohort is drawn from a predominantly high
socioeconomic population in a developed country and therefore the
results may not be generalizable to children in developing
countries with lower socioeconomic status where the NP colonization
frequency and bacterial load may be higher.
[0344] In conclusion, this is the first study to compare antibody
levels to three NTHi candidate vaccine OMPs in children following
asymptomatic NP colonization and episodes of AOM. Increasing levels
of protein D, P6 and OMP26-specific IgG antibodies were found in
children as they increased in age from 6 to 30 months of age.
Increased antibody levels were specifically measured following
detected NP colonization with NTHi for protein D and P6 but not
OMP26. In convalescence from AOM children had lower overall IgG
antibody levels than after asymptomatic NP colonization. Thus it
appeared that AOM occurred in the context of a less robust immune
response than following colonization. However the overall response
did not reflect individual responses among the study cohort in that
some children had a clear increase in antibody to protein D, P6
and/or OMP26 following AOM. Natural antibodies to protein D and P6
but not OMP26 elicited by NP colonization and AOM were
bactericidal.
F. Example 6
Reduced Serum IgG Responses to Pneumococcal Antigens in Otitis
Prone Children May be Due to Poor Memory B-Cell Generation
1. Introduction
[0345] Streptococcus pneumoniae (Spn) is one of the most common
pathogens causing AOM [Casey et al., Pediatr. Infect. Dis. J. 2010;
29(4):304-9]. Studies in animal models, and in humans to some
extent, suggest that immune correlates of protection from infection
by Spn include memory CD4.sup.+ T cells, B cells, neutralizing
serum and mucosal antibody levels [Zhang et al., J. Infect. Dis.
2007; 195(8):1194-202; Snapper et al., Trends Immunol. 2001;
22(6):308-11; Weiser et al., Proc. Natl. Acad. Sci. U.S.A 2003;
100(7):4215-20]. It was recently established that otitis prone
children have reduced frequencies of Spn and nontypeable
Haemophilus influenzae (NTHi)-antigen-specific memory CD4.sup.+ T
cells in their circulation at the time of AOM and following
nasopharyngeal (NP) colonization [Sharma et al., J Infect Dis.
2011; 204(4):645-653]. After natural infection and vaccination,
robust memory T and B cell responses should be generated, with
memory lymphocytes populating lymphoid and non-lymphoid sites, to
provide long-term protection from re-infection [Pichichero,
Pediatrics 2009; 124(6):1633-41]. Once generated on subsequent
exposure to a pathogen, memory B cells can proliferate into
antibody secreting cells (ASCs) and maintain serum antibody levels
over a period of time [Lanzavecchia and Sallusto, Curr. Opin.
Immunol. 2009; 21(3):298-304; Kelly et al., JAMA 2005;
294(23):3019-23].
[0346] Earlier reports describe that otitis prone children produce
lower amounts of Spn and NTHi-antigen-specific antibodies and/or
not to produce functional bactericidal antibodies in response to
AOM and/or NP colonization [Faden, Eur. J. Pediatr. 2001;
160(7):407-13; Murphy and Yi, Ann. N.Y. Acad. Sci. 1997;
830:353-60; Kaur et al., Vaccine 2011; 29(5):1023-8]. These
findings indicate that decreased concentrations of circulating
antibodies to the otopathogens may contribute to the otitis prone
condition. However, until this current work there has not been an
evaluation of whether the observed reduction in the serum antibody
in otitis prone children might be due to failure to generate robust
antigen-specific memory B cells. This is the first report
demonstrating that lower pathogen-specific memory B cell generation
may account for lower antibody levels to protein antigens displayed
by Spn among young children from recurrent episodes of AOM.
2. Methods
[0347] i. Subjects
[0348] Subjects were participants from our 5-year prospective
longitudinal AOM study funded by the NIH NIDCD [Kaur et al.,
Vaccine 2011; 29(5):1023-8]. Enrolled children were from a middle
class, suburban socio-demographic population in Rochester N.Y.
Healthy children at age of 6 months without prior AOM were enrolled
and had blood, NP and oropharyngeal (OP) cultures obtained seven
times, at the age of 6, 9, 12, 15, 18, 24 and 30 months. Middle ear
fluid (MEF) was obtained by tympanocentesis during AOM episodes.
Colonization with Spn and/or NTHi in the NP/OP was routinely
determined by standard microbiologic culture. To identify the
otitis prone child in the study population all the children had
tympanocentesis-confirmed infections and all received antibiotic
therapy directed to the otopathogen isolated from middle ear fluid
for each AOM event. PBMCs were isolated from the collected blood
and frozen in the liquid nitrogen until used. Children having three
episodes of AOM within 6 months or 4 episodes within one year were
considered otitis prone while others who had fewer episodes were
placed into the non-otitis prone group. Written informal consent
was obtained in association with a protocol approved by the
Rochester General Hospital Investigational Review Board.
ii. Antigens
[0349] Five different pneumococcal protein antigens were used in
this study: pneumococcal histidine triad proteins D (PhtD) and E
(PhtE), LytB, PcpA, PlyD1 (a detoxified derivative of pneumolysin
which has three point mutations that do not interfere with
anti-pneumolysin antibody responses). All these antigens are
pneumococcal vaccine candidate antigens.
iii. Humoral Responses
[0350] For measuring IgG antibody levels in the samples, ELISA was
performed as described previously [Kaur et al., Vaccine 2011;
29(5):1023-8]. Briefly, 96-well ELISA plates (Nunc-Immulon) were
coated with 0.5 ug/ml of individual antigens (100 .mu.l/well) in
coating buffer (bicarbonate, [pH 9.4]) and incubated overnight at
4.degree. C. After washing, the plates were blocked with 3% skimmed
milk at 37.degree. C. for 1 hr (200 .mu.l per well). After five
washes, 100 .mu.l of serum at a starting dilution of 1:100 (in
PBS-3% skim milk) was added to the wells and diluted serially 2
fold. The mixture was incubated at room temperature for 1 hr
followed by the addition of affinity purified goat anti-human IgG,
IgM or IgA antibody conjugated to horseradish-peroxidase (Bethyl
Laboratories, Inc, Montgomery, Tex.) as a secondary antibody. The
reaction products were developed with TMB Microwell Peroxidase
Substrate System (KPL, Gaithersburg, Md.), stopped by the addition
of 1.0 molar phosphoric acid and read by an automated ELISA reader
using a 450-nm filter. To provide quantitative results on antibody
concentrations, the level of the specific antibody present in the
unknown sample was determined by comparison to an internal
reference serum (pool of human serum with high antigen titers). The
levels of IgG in the reference serum were quantitatively measured
by using a human IgG ELISA quantitation kit (Bethyl laboratories).
A Four-parameter logistic-log function was used to form the
reference and sample curves.
iv. Antibody Secreting Cells (ASCs) ELISPOT
[0351] Antigen-specific as well as total IgG secreting cells were
quantified by an assay in which memory B cells were stimulated in
vitro to differentiate into antibody-secreting cells (ASC) as
standardized in the laboratory. Briefly, one million thawed PBMC
were placed in each well of a 24-well plate containing 1 ml of
complete media alone or complete media containing 1 .mu.g/ml of
pokeweed mitogen. Cells were kept at 37.degree. C. for 3-days for
differentiation, washed with complete media, counted and
distributed onto overnight antigen-coated (10 .mu.g/ml) 96-well
ELISPOT plates (Millipore). Plasma cell differentiation was
optimized with the help of flow cytometric evaluation of the
differentiated cells (data not shown). For the detection of total
IgG-secreting cells, wells were pre-coated with monoclonal
anti-human IgG (MT91/145; Mabtech) at 10 ug/ml in PBS. As a
negative control wells were left untreated or coated with same
amount of bovine serum albumin (BSA). Plates were blocked with 10%
FBS in RPMI 1640 for 30 min at 37.degree. C. Stimulated PBMC were
counted and 5.times.10.sup.5 cells were resuspended in 200 .mu.l of
fresh complete RPMI media before distributing them onto control and
antigen-coated wells. Plates were then incubated at 37.degree. C.
in a 5% CO.sub.2 incubator overnight and then washed with PBS at
least 5-times. Next, 100 .mu.l of 1 .mu.g/ml biotinylated
anti-human IgG antibodies (MT78/145; Mabtech) were added to the
wells and incubated for an hour. After washing
streptavidin-alkaline phosphatase conjugate (1:1000) was added to
the wells and incubated for an hour at 37.degree. C. Plates were
then washed 5-times with PBS before developing it with substrate
(BCIP/NBT; Mabtech). Because of the low frequencies of
antigen-specific ASCs, developed spots were manually counted with
the help of dissection microscope. Ag specific data was expressed
as a percentage of antigen-specific memory B-cells and was
calculated per million of PBMC as follows: % Ag-specific MBC=(No.
antigen-specific spots/No. of total Ig spots).times.100.
v. Statistics
[0352] All data was analyzed using Graph Pad Prism software. Two
tailed P values for the data were calculated using Mann Whitney
Test.
3. Results
[0353] i. Study Population
[0354] From a total study population of 387 children otitis prone
children were identified. From the remainder children with 1 or 2
AOMs who were of a similar age as the non-otitis prone children
were randomly selected to serve as controls. Clinical
characteristics of the study children are shown in Table 5.
TABLE-US-00010 TABLE 5 Characteristics of study subjects Otitis
Prone Non-Otitis Prone (n = 10) (n = 12) P value Gender Male 6 7
1.00 Female 4 5 1.00 Mean Age (mos) 13.3 12.1 0.50 #AOM Episodes
.gtoreq.3 in 6 months 5 0 0.01 .gtoreq.4 in 12 months 5 0 0.01
Total number of AOM Episodes 1-3 3 4 1.00 4-5 6 0 0.003 6 or more 1
0 0.45 PET Insertion 4 0 0.03 Breast Feeding .gtoreq. 6 months 5 8
0.67
ii. Generation of Pneumococcal Antigen-Specific Memory B-Cell is
Reduced in Otitis Prone Children
[0355] The circulating frequencies of various Spn antigen-specific
memory B cells were compared between non-otitis prone and otitis
prone children by stimulating their PBMCs with polycloncal
stimulation. Antigen specific B cell responses were normalized with
the control ELISPOT plate wells left uncoated or coated with
BSA.
[0356] FIG. 18A demonstrates percentages of memory B cells to 5-Spn
antigens in otitis prone children and non-otitis prone children
caused by Spn. In sharp contrast, otitis prone children had a
marked reduction of circulating Spn specific memory B cells after
their AOM or NP colonization (Table 5). In particular,
significantly lower percentages of memory B cells producing
antigen-specific IgG were observed against antigens PhtD, PhtE and
PlyD1 (P<0.02). Although otitis prone children showed an overall
lower memory B cells generated to LytB, however the difference was
not found significant (p=0.1). No difference was found in the
memory B cells to PcpA in both otitis prone and non-otitis prone
children (FIG. 18A). Similarly, total IgG-secreting cells were not
different among both of the groups (data not shown).
iii. Otitis Prone Children have Reduced IgG Concentration to
Pneumococcal Protein Antigens
[0357] Antigen-specific IgG titers were evaluated in the serum of
otitis prone and non-otitis prone children of matching age group.
Serum IgG levels to Spn antigens in the respective groups are shown
in FIG. 18B. In the cohort of non-otitis prone children IgG titers
to PhtD, PcpA and PhtE were significantly higher compared to otitis
prone (P<0.05), whereas PlyD1 levels were lower and not
significantly different between the groups (FIG. 18B). Antibodies
to LytB were lowest among all antigens tested in both of the
cohorts (FIG. 18B).
4. Discussion
[0358] In this study, it was found that a reduced percentage of
memory B cells circulating in the blood of otitis prone children
following AOM and/or NP colonization (FIG. 18A). After encounter of
antigen with naive B cells, antigen-specific memory B cells and
antibody secreting cells are generated in the secondary lymphoid
structures that transit through the blood to bone marrow, spleen,
or target tissues such as respiratory tract [Kelly et al., JAMA
2005; 294(23):3019-23]. Since serum antibody levels are maintained
by memory B cells [Bernasconi et al., Science 2002;
298(5600:2199-202], by analyzing the percentages of generated
antigen-specific memory B cells a more precise immunological
explanation for lower antibody levels in otitis prone children was
provided. To confirm the association of lower frequencies of memory
B cells with serum antibody levels we measured Spn-specific
antibody titers and found they were significantly lower in otitis
prone children (FIG. 18B).
Recently, it was demonstrated that otitis prone children have
suboptimal pneumococcal antigen-specific memory CD4.sup.+ T cell
responses [Sharma et al., J Infect Dis. 2011; 204(4):645-653].
Findings from this study indicate that otitis prone children may
develop some antibody responses since antibodies and memory B cells
were detectable among these children after AOM and NP colonization
with otopathogens (FIG. 18A-B). However, in the absence of
antigen-specific memory B cell generation as well as adequate help
from memory CD4.sup.+ T cells, the antibody levels wane and otitis
prone children are unable to maintain adequate serum antibody
levels and get frequent repeat infections.
[0359] Pneumococcal polysaccharide-conjugate vaccination is helpful
in boosting protective levels of anti-polysaccharide antibodies
[Barnett et al., Clin. Infect. Dis. 1999; 29(1):191-2]; however
serotype variation limits the protective efficacy of strain
specific anti-polysaccharide antibodies [Casey et al., Pediatr.
Infect. Dis. J. 2010; 29(4):304-9]. Moreover, despite of the fact
that otitis prone children can induce serotype specific antibodies
to conjugate vaccines, repeated infections are common among this
vulnerable group [Barnett et al., Clin. Infect. Dis. 1999;
29(1):191-2], indicating that serotype-neutralizing immunity is
brief and incomplete.
[0360] Interestingly, it was found that the percentage of
circulating PhtD specific memory B-cells correlated with serum PhtD
levels (FIG. 18C). A difference in the percentages of
antigen-specific B cells and serum antibodies levels to PcpA and
PlyD1 was observed (FIG. 18A-B). It is possible that (1) by binding
to the circulating IgG, an active state of NP colonization or AOM
infection may affect the detection of serum antibody levels as
opposed to memory B cells, and (2) during infection in the
uncontrolled inflammatory environment of NP, a different dose of
pathogen antigen and PAMPs stimulation may elicit variable
frequencies of B cell differentiation into ASCs and thus affect
serum IgG levels even in the presence of memory B cells.
[0361] In conclusion, the memory B cell data indicate that otitis
prone children have a significantly lower memory B cell generation
that can differentiate into antibody secreting cells. The clinical
relevance of the finding is clear. Antigen specific memory B cells
act as reservoirs for serum antibody maintenance that upon antigen
re-encounter can proliferate into ASCs leading to an increase in
the serum antibody levels. It was found that otitis prone children
do not lack total IgG-secreting cells. Furthermore the flow
cytometry results showed that in response to polyclonal
stimulation, otitis prone children do not have mechanistic
dysfunction in the transformation of memory B cells (CD19+IgD-) to
antibody secreting plasma-cells (CD27+CD38+CD138+) (data not
shown). Whether naive B cells in the secondary lymphoid organs of
otitis prone children are unable to get optimal CD4.sup.+ T-cells
or T-follicular cell help for differentiation into memory B cells
and/or ASCs for eventually maintaining higher serum IgG levels is
currently being investigate.
Sequence CWU 1
1
11153PRTHaemophilus influenzae 1Met Asn Lys Phe Val Lys Ser Leu Leu
Val Ala Gly Ser Val Ala Ala1 5 10 15Leu Ala Ala Cys Ser Ser Ser Asn
Asn Asp Ala Ala Gly Asn Gly Ala 20 25 30Ala Gln Thr Phe Gly Gly Tyr
Ser Val Ala Asp Leu Gln Gln Arg Tyr 35 40 45Asn Thr Val Tyr Phe Gly
Phe Asp Lys Tyr Asp Ile Thr Gly Glu Tyr 50 55 60Val Gln Ile Leu Asp
Ala His Ala Ala Tyr Leu Asn Ala Thr Pro Ala65 70 75 80Ala Lys Val
Leu Val Glu Gly Asn Thr Asp Glu Arg Gly Thr Pro Glu 85 90 95Tyr Asn
Ile Ala Leu Gly Gln Arg Arg Ala Asp Ala Val Lys Gly Tyr 100 105
110Leu Ala Gly Lys Gly Val Asp Ala Gly Lys Leu Gly Thr Val Ser Tyr
115 120 125Gly Glu Glu Lys Pro Ala Val Leu Gly His Asp Glu Ala Ala
Tyr Ser 130 135 140Lys Asn Arg Arg Ala Val Leu Ala Tyr145 150
* * * * *