2.0 of fresh BHI media and shaken at

2.0 Materials and methods

2.1 Ethics statement and animals

All
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
determination

A
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

A
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
Assay

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

Transformed
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

Each
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
conjugates

Conjugates
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<0.05) was determined using an unpaired Mann-Whitney U test to compare between test groups (p<0.05 was considered significant) using GraphPad Prism 6 software (GraphPad, California, United States). 2.8 Opsonophagocytosis assay The antibodies raised against respective LOS to opsonize M. catarrhalis and there by promote phagocytosis of the bacteria was developed with reference to similar assays used to analyse opsonization of other species of bacteria (29–33). M. catarrhalis 2951 wild-type, 3292 wild-type, 2951lgt1/4? and NTHi strain 285 was grown overnight as mentioned above, harvested, washed twice in sterile PBS and fluorescently dyed with 5-chloromethylfluoresceindiacetate (CMFDA) using the method suggested by the manufacturer (Sapphire Bioscience). Approximately 1×1010 CFU was harvested from agar plates, washed in PBS, and resuspended in 400 ?L of 5 ?M Chemodex Green CMFDA diluted in BHI media. The bacteria were incubated for 30 min at 37?C, pelleted again by centrifugation, resuspended in 1 mL BHI media, and again incubated for 30 min at 37?C. The bacteria were washed five times in Hank's buffered salt solution (HBSS) with 0.2% BSA and after the final wash their concentration was adjusted to 2×108 CFU/mL by estimation from the OD400. Twenty microliters of the bacterial suspension were mixed with 10 ?L of mouse serum and incubated for 30 min at 37?C with gentle shaking (150 rpm). The serum samples were diluted to approximately the same relative antibody concentration as determined by ELISA (approximately 20 ?g/mL) so that the results would reflect the opsonizing ability of the antibodies rather than to differences associated with titer to the specific antigens. Ten microliter of guinea pig serum or rabbit serum was added as an exogenous complement source and the mixture was incubated for a further 15 min at 37?C with gentle shaking. Mouse macrophages (RAW 264.7) were washed twice with HBSS and once with HBSS containing Ca2+ and Mg2+ and the concentration of cells was adjusted to 2.5×106 cells/mL. Forty microliters of this macrophage suspension were added to the bacterial suspension. The mixture was incubated for 30 min at 37?C with gentle shaking to allow phagocytosis of the bacteria by the macrophages. The cells were diluted with 200 ?L of HBSS with Ca2+ and Mg2+ with 0.2% BSA, 50 ?L of 0.5 mg/mL trypan blue was added to quench the fluorescence of extracellular bacteria and the cells were kept on ice before analysis using a flow cytometer (Beckman Coulter XL-MCL). The relative fluorescence of the macrophages was used to assess the degree of phagocytosis in each sample. A higher percentage of fluorescent macrophages indicated a higher percentage of these cells had phagocytosed bacteria. Controls were included that received buffer instead of the mouse serum and serum from non-immunized mice and mice immunized with whole killed M. catarrhalis 2951 and 3292 (WKC) were included as negative and positive controls, respectively. 2.9 Bactericidal assay The bactericidal activity of antibodies raised against the conjugates and OMP26 was assessed using the method described by Murphy et al.(34). Serum samples from subcutaneously immunized mice were heat-inactivated by incubation at 56?C for 30 min before use. The serum was then diluted with PBS to give an equivalent concentration of antibody as determined by ELISA. M. catarrhalis strains CCUG 2951 (serotype A; LOS source strain), CCUG 3292 (serotype B; LOS source strain), ATCC 25238 (serotype A; LOS source strain), ATCC 25239, ATCC 26397 (serotype B; LOS source strain), ATCC 26400 and ATCC 26404 (serotype C; LOS source strain) was grown on BHI agar plates at 37?C under 5% CO2 overnight. Few colonies from plate was inoculated into BHI broth and incubated overnight 37?C with shaking at 220 rpm and from the overnight culture an inoculant was grown in BHI broth at 37?C with shaking at 220 rpm until the OD600 was approximately 0.2. An aliquot of the culture was diluted with sterile Dulbecco's PBS (calcium, magnesium and 0.1% gelatin) and plated to determine CFU, to adjust bacteria to 103 CFU. Thirty microliters of this cell suspension (approximately 100-200 CFU per well for convenient counting) was added to each well of the microtiter plate. Ten microliter of heat-inactivated serum sample was added to each well, with each serum sample being tested in triplicate. Serum from mice immunised with whole heat killed M. catarrhalis 3292 and 2951 cells was included as a positive control. Control wells without serum received sterile PBS instead of serum. The samples were mixed gently by pipetting and incubated for 30 min at room temperature to allow the antibodies present in the mouse serum to bind to the bacteria. Ten microliter of rabbit serum was then added to each well as the complement source and the cells were incubated for a further 30 min at 37?C to allow complement mediated killing of the bacteria. Wells that were not complement controls received sterile PBS instead of rabbit serum. Two hundred microliter of sterile DPBS was added to each well and 20 ?L aliquots were spotted on to BHI plates in duplicate to enable quantification of live bacteria. The plates were incubated overnight and the number of colony forming units in each spot was counted. The bacterial counts were compared with the negative control wells that did not have mouse serum, complement source or both. Serum from non-immunised mice was also included as a negative control. A positive result is considered killing that reduces recovery to <50% of the recovery from the negative control. 2.10 Limulus amebocyte lysate assay The LOSs and dLOSs were tested by the ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GeneScript, New Jersey, United States). The sensitivity of the LAL assay is between 0.005-1 endotoxin unit (EU)/?L. For whole cell endotoxin activity, overnight bacterial culture from BHI agar plates were suspended in BHI broth to a OD600 of 0.1 and serial dilutions were tested with the kit (2).