2.0 of fresh BHI media and shaken at

2.0 Materials and methods

2.1 Ethics statement and animals

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animal protocols were reviewed and approved by the Griffith University Animal
Ethics Committee in accordance with the National Health and Medical Research
Council of Australia guidelines (AEC protocol number: MSC/11/15). Female BALB/c
mice were (four-to-six-week-old) were sourced from the Animal Resource Centre
(Perth, WA, Australia).

2.2 Bacterial strains and culture conditions

M. catarrhalis strains were cultured in brain heart infusion
(BHI; Oxoid, Heidelberg, Victoria, Australia) agar at 37°C overnight. M. catarrhalis 2951lgt1/4? mutant strain
was grown on BHI agar supplemented with 20 µg.mL-1 kanamycin. The
bacteria were pelleted by centrifugation at 3,000 x g. For disc diffusion
assays, M. catarrhalis were cultured
in BHI broth to a OD600 of 0.2 and 100 µL of the bacterial
suspension spread (lawn cultured) onto BHI agar plates. The growth rate profile
of strains was measured as follows: an overnight liquid culture was inoculated
(1:50 inoculation) into 100 mL of fresh BHI media and shaken at 37°C at 250
r.p.m., with spectrophotometer monitoring at OD600 every 30 mins
until readings become steady. The growth rate was measured in triplicates, on
three separate days and the average plotted as a curved graph. NTHi-289 was grown in BHI agar plates and
broth at 37°C in CO2 supplemented with NAD and hemin. Transformed E. coli pop1235 containing the outer
membrane protein 26 gene was obtained from Dr Ian Peak and grown in
Luria-Bertani (LB) agar and LB broth supplemented with 100 µg/mL of ampicillin.

2.3 Biological activity of the LOS
truncated mutant

2.3.1 Susceptibility

standard disk-diffusion assay was performed using a panel of hydrophobic agents
(vancomycin (30 µg), Tween 20 (5%, w/v), Triton X-100 (5%, w/v), rifampin (5
µg), polymixin B (300 international units), novobiocin (5 µg), chloramphenicol
(10 µg) and nalidixic acid (30 µg)). Antibiotic disks were used to plate
various agents on the lawn cultured plates for 18 hours at 37°C. The
sensitivity was reported as size of growth inhibition and the assay was tested
in triplicate (2).

2.3.2 Bactericidal
assay with normal human serum

bactericidal assay was conducted in a 96 well plate, with a final volume of 100
?L. Normal human serum in triplicates was diluted to 0.5, 2.5, 5.0, 12.5 and
25% in pH 7 Dulbecco’s phosphate buffered saline containing 0.05% gelatin
(DPBSG). Inoculation of bacterial culture (10 ?L of 106 CFU) was
carried out into reaction wells containing the diluted bovine serum and 25% of
heat-inactivated (65°C) normal serum, and incubated for 30 minutes
at 37°C. Serial dilutions (1:10) were plated in triplicates and CFU counted
after 24 hrs (2).

2.3.3 Adherence

HeLa cervix and Chang conjunctival human cell lines were
cultured in Eagle’s minimal essential medium with addition of 10% heat
inactivated fetal bovine serum at 5% CO2 at 37°C. Approximately 2×105
to 3×105 HeLa and Chang cells were seeded into each well of a 24
well tissue culture plate (Corning-Costar) and incubated for 24 hrs before use.
Bacterial colonies were inoculated into 10 mL of BHI broth and grown overnight
at 37°C. The overnight culture was pelleted by centrifugation at 6,000 x g for
10 mins at 4°C, washed twice with 5 mL of phosphate-buffered saline (PBS), pH
7.0, and resuspended in 10 mL of PBS. Bacteria was then inoculated (25 µL of 107
CFU) in triplicates onto the wells of the tissue culture plate containing
monolayers and centrifuged for 5 min at 165 x g and then incubated for 30 min
at 37°C. The infected monolayers were rinsed five times with PBS to remove
nonadherent bacteria and then treated with PBS containing trypsin-EDTA (0.05%
trypsin, 0.5 mM EDTA) to release the epithelial cells from the plastic support.
This cell suspension was serially diluted in PBS and spread onto BHI agar plates
in duplicates to determine the number of viable M. catarrhalis present. Adherence of the bacteria
attached to human cells relative to original bacteria added, was expressed as a
percentage (2,20)

2.4 Conjugate vaccine

2.4.1 NTHi outer membrane protein 26 expression

E. coli with the plasmid pQE30
containing the omp26 gene derived from NTHi
strain 289 was grown as described above. The recombinant E. coli strain was cultured at 37°C until OD600
was approximately 0.6-0.7. Isopropyl ?-D-thiogalactopyranoside (1 mM) was added
to induce expression of OMP26 antigen, controlled by LacPO promoter and further
incubated for 4-6 hrs. The cells were harvested at 5,700 rpm centrifugation at
4°C for 20 min. The pellet was resuspended in
lysis buffer at a wet weight of 5 mL/g. The cells were cracked open by
incubation with lysozyme on ice for 30 min, followed by sonication. The lysed
slurry was then centrifuged (8,000 rpm, 20 min, 4°C) and
supernatant removed (6) and purified using a Ni-NTA Fast Start
(QIAGEN, Hilden, Germany). The purity of OMP26 was analysed using SDS-PAGE
(Sigma-Aldrich, Chemical
Co., Castle hill, Australia). Protein
concentration was determined with the Pierce Bovine serum albumin assay kit
(ThermoFisher Scientific, Australia).  

2.4.2 M. catarrhalis wild-type and mutant LOS isolation, detoxification
and derivatization

M. catarrhalis LOS was extracted from 5-10 g of wet
cells from 2951 and 3292 wild-type and 2951lgt1/4? using the hot phenol-water procedure
by Westphal and Jann (21), with minor modifications by Perdomo
and Montero (22) and Wilson et al (). Each crude LOS (200 mg) from 2951 wild-type, 3292
wild-type, and 2951lgt1/4? was detoxified with ammonium
hydroxide and purified using Sephadex G-50 (). The
final carbohydrate-containing fractions were freeze-dried, analysed on 1H
NMR and designated as detoxified LOS (dLOS).
Each dLOS (60 mg) was derivatized
with a linker, adipic acid dihydrazide (ADH; Sigma-Aldrich Chemical Co., Castle
hill, Australia) (23). The purified derivative containing
both carbohydrate and adipic hydrazide (AH) was lyophilized and named as AH-dLOS.

2.4.3 Conjugation of AH-dLOS to protein

AH-dLOS (30 mg) was coupled to OMP26 (1:100)
to form conjugates as described (1). The purified compound containing both
carbohydrate and protein was pooled and designated as dLOS-OMP26. All conjugates were analysed for their composition of
carbohydrate and protein, using AH-dLOS
and bovine serum albumin (BSA) as standards (24,25).

2.5 Subcutaneous immunisation of mice with

were tested for immunogenicity in mice. Four to six-week-old female BALB/c mice
(eight per group) were immunised subcutaneously on days 0, 14 and 28 with 50 ?g
of 2951lgt1/4dLOS-OMP26 delivered with
Ribi adjuvant (Sigma-Aldrich
Chemical Co., Castle hill, Australia)
at a 1:1 ratio (100 ?L immunisation dose/mouse); 2951dLOS-OMP26 (50 ?g); 3292dLOS-OMP26
(50 ?g); OMP26 (50 ?g), heat-killed (65°C for 30 mins)
2951 wild-type strain (100 ?L of 106 CFU/mouse) and heat-killed 3292
wild-type strain.

Serum was collected on days -1, 13, 27 and 42 before each
immunisation to determine the level of dLOS
and OMP26-specific systemic antibodies. Blood was collected from the mice via
the vein slightly behind the mandible and allowed to clot overnight. Serum was
collected after centrifugation for 10 min at 3000 g and stored at -80°C.

2.6 Whole-cell enzyme-linked immunosorbent assay (ELISA)

M. catarrhalis strains 2951 and 3292 were grown in BHI broth
to an OD600 of 0.2, harvested by centrifugation, and resuspended in
PBS. A volume of 100 ?L of the suspension was added to each
well of the 96-well Titertek PVC microplates (MP biomedicals) (26).
Wells with PBS alone and day -1 pre-sera was included as
controls. The rest of the experiment was performed according to the protocol
described below for ELISA with respective LOS and OMP26.

2.7 Determination
of antibody titers by ELISA

ELISA was used to measure serum IgG against the
conjugates, OMP26 and whole cell bacteria, as described elsewhere (27,28) . Briefly, respective LOS and OMP26 was
diluted to 1 mg/mL in carbonate coating buffer, pH 9.6, and coated onto
Titertek PVC microplates (MP biomedicals) in a volume of 100 ?L/well overnight
at 4°C or 1.5 h at 37°C. Unbound peptide was removed and each well was blocked
with 150 ?L of 5% skim milk PBS overnight at 4°C or 1.5 h at 37°C. The plates were then washed 3 times with PBS. Serum samples were assessed using 2-fold dilutions
of 1:100 dilution of serum. Each sample was diluted to a final volume of 100 ?L
and incubated for 1.5 h at 37°C. heat killed, respective LOS or OMP26 mouse
antibodies were detected with HRP-conjugated goat anti-mouse IgG antibody
(Bio-rad Laboratories), HRP-conjugated goat anti-mouse IgG1 antibody
(Invivogen, San Diego, United States), HRP-conjugated goat anti-mouse IgG2a
antibody (Invivogen, San Diego, United States), HRP-conjugated goat anti-mouse
IgG2b antibody (Invivogen, San Diego, United States) or HRP-conjugated goat
anti-mouse IgG3 antibody (Invivogen, San Diego, United States) added at a
dilution of 1:3000 or 1:2000 respectively in 0.5% skim milk PBS for 1.5 h at
37°C. SIGMAFAST OPD substrate (Sigma Aldrich) was added at 100 ?L per well
according to manufacturer’s instructions and incubated at room temperature for
20 min in the dark. The absorbance was measured at OD450 in a Tecan Infinite
M200 Pro plate reader (Tecan Group Ltd., Switzerland). The titer was
described as the lowest dilution that gave an absorbance of >3 standard
deviations (SD) above the mean absorbance of negative control wells (containing
day -1 pre-sera from mice). Statistical significance (p