Adjuvants and antigens
PapMV nanoparticles (lot # Eng-1211) were provided by Folia Biotech Inc. (Quebec City,
Quebec, Canada) and were produced as described in our previous study 21]. In brief, PapMV nanoparticles used in this report are self assembled in vitro with
a non-coding ssRNA in vitro transcript of 1517 nucleotides. The nanoparticles show
an average length of 100 nm and a diameter of 15 nm as shown by electron microscopy
(Additional file 5: Figure S1A) and dynamic light scattering (Additional file 5: Figure S1B). The potential zeta was estimated at ?6.12 mV (Additional file 5: Figure S1C), and is consistent with other batches of PapMV nanoparticles produced
in the laboratory. LPS contamination was always below 50 endotoxin units (EU)/mg of
protein, and was considered negligible.
The commercially available formulation of the trivalent inactivated influenza vaccine
(TIV) 2013–2014 (lot #3YF27) (A/California/7/2009 NYMC X-179A (H1N1), A/Texas/50/2012
NYMC X-223A (H3N2) and B/Massachusetts/2/2012 NYMC BX-51B) from GlaxoSmithKline (GSK;
Fluviral) was used as the model antigen.
Mice immunization and depletion
The PapMV nanoparticles were formulated in 10 mM Tris pH 8.0 at a concentration of
0.5 mg/mL. TIV was prepared by GSK and contained 15 µg of each of the HA in a volume
of 0.5 mL. Seven- to ten-week-old female BALB/c mice were immunised once by i.m. injection
of 50 µL of a formulated vaccine, 1 µg (of each hemagglutinin) TIV+ 15 µg of PapMV
nanoparticles, 1 µg (of each hemagglutinin) TIV or buffer (10 mM Tris pH8.0). Formulations
were prepared and administered on the same day with syringes with 30G × 5/16? Needles.
All the i.m. injection were in a final volume of 50 µL.
CD4 depletion was performed using 200 µg of rat anti-CD4 (Clone GK1.5) (Biolegend,
San Diego, CA, USA) injected intraperitoneally (i.p.). Depleted mice were immunised
1 day post-depletion. Depleted mice were assayed for remaining CD4+ cells in the blood
by flow cytometry, and immunised 1 day post-depletion. Cells were washed and stained
with rat anti-CD4 (Clone GK1.5) (Biolegend, San Diego, CA, USA) and Alexa Fluor
®
488 goat anti-rat IgG (LifeTechnologies, Burlington, Ontario, Canada). Cells were
analyzed by flow cytometry using a BD FACS Canto flow cytometer (BD, Mississauga,
Ontario, Canada). The approval of this project for animal work is found under authorization
number 2013-142 of the “Comité de Protection des Animaux-CHUQ (CPA-CHUQ)”.
Antibody assay
Blood samples were collected from mice and centrifuged in BD microtainer blood collection
tubes (BD, Mississauga, Ontario, Canada) for 2 min at 10,000×g. Considering ethical obligations and blood sampling limitations, each time point
is represented by a different group of five mice. The serum were assayed for total
IgG, IgG2a serotype, and IgM against TIV and IgG2a against GST-fused influenza nucleoprotein
(GST-NP) by enzyme-linked immunosorbent assay (ELISA) as described elsewhere 15], 21]. IgGs ELISA were conducted by serial dilutions in twofold steps starting at 1 in
50 of serum in dilution buffer. Results are expressed as an antibody endpoint titer
greater than threefold the background optical density values consisting of preimmune
sera. IgM titers were measured by the optical density values of sera with a dilution
factor 1:400.
B cells enzyme-linked immunospot
Enzyme-linked immunospot (ELISPOT) 96-well PVDF-membrane microtiter plates (Millipore,
Billerica, MA, USA) were activated with 50 µL of 35 % ethanol for 30 s, and washed
five times with phosphate-buffered saline (PBS). Plates were then coated overnight
with 2 µL of TIV in 100 µL PBS. Wells were then washed five times and blocked for
30 min with RPMI 1640 medium containing 10 % decomplemented foetal bovine serum. Mice
lymph nodes were extracted at days 5 and 14 post-immunization and digested with type
IV Collagenase (0.5 mg/mL) and DNase I (0.125 mg/mL) at 37 °C for 30 min. Digested
lymph nodes were transferred into a 50 mL tube through a 70 µm mesh and washed with
PBS +2 mM EDTA. Cells were then centrifuged at 500×g for 5 min at 4 °C and the supernatant was discarded. Lymph node cells (1 × 105) were
deposited in the coated wells and incubated for 18 h at 37 °C, 5 % CO
2
. Antibodies were detected using a biotinylated anti-IgG (Life technologies, Burlington,
Ontario, Canada) at 1 µg/mL and a streptavidin–alkaline phosphatase (Promega, Madison,
WI, USA) diluted 1:1000. Spots were revealed with 100 µL of BCIP-NBT for 15 min, and
then washed with distilled water. Plates were observed using a SMZ800 optical microscope
(Nikon, Mississauga, Ontario, Canada) and counted automatically using ImageJ (Version
1.43 m, NIH, USA).
Germinal center assay by flow cytometry
The lymph nodes (5 mice per group) were extracted at days 5 and 14 post- immunisation
and digested with type IV Collagenase (0.5 mg/mL) and DNase I (0.125 mg/mL) at 37 °C
for 30 min. Cells were stained with anti-mouse-CD45R-PerCP (0.2 µg, BD, Mississauga,
Ontario, Canada), lectin PNA from Arachis hypogaea-AlexaFluor 647 (1 µg, Life technologies, Burlington, Ontario, Canada) and anti-mouse
T- and B-cell activation antigen-FITC (Clone GL7) antibody (0.2 µg, BD, Mississauga,
Ontario, Canada). Staining was done for 30 min at 4 °C and cells were then washed
3 times with 1 mL of cold flow cytometry buffer. Germinal centers were assayed on
a flow cytometer by first gating for positive CD45R staining. Cells were considered
to originate from GCs when they stained highly for PNA and GL7.
Frozen section and confocal microscopy
Mice inguinal lymph nodes from the same side as the immunization site were extracted
at day 14 and placed in RPMI 1640. Lymph nodes were then transferred into a mold containing
premium frozen section compound (OCT) (VWR, Ville Mont-Royal, Quebec, Canada). Lymph
nodes were then covered by OCT and snap-frozen in liquid nitrogen. 8-µm-thick sections
were prepared using a Jung Cryostat 2800 Frigocut-E (Leica, Concord, Ontario, Canada)
and applied to positively-charged slides. Sections were fixed for 20 min using 4 %
paraformaldehyde in PBS and rinsed. Sections were then stained with anti-CD45R-AlexaFluor
488 (Clone RA3-6B2, Biolegend, San Diego, CA, USA) and PNA-AlexaFluor 647 (Life technologies,
Burlington, Ontario, Canada) in staining buffer (Phosphate buffer + 5 % bovine serum
albumin). Slides were mounted using SlowFade gold antifade mountant (Life technologies,
Burlington, Ontario, Canada) and observed with a Quorum WaveFX (Quorum Technologies,
Guelph, Ontario, Canada) confocal spinning disk microscope at 10×.
Challenge with a heterologous influenza virus
Immunised mice, 10 per group, were challenged with 1.5 LD50 of A/WSN/33 (H1N1) influenza
viruses at different time points post-immunization by an intranasal instillation of
50 µL viral preparation following anesthesia by isoflurane. Mice were monitored for
14 days for survival. Mice that lost 20 % or more of their initial weight were euthanized.
Mice immunized with TIV and infected with A/WSN/33 (H1N1) develop viral symptoms and
are not protected against a mild challenge. A/WSN/33 (H1N1) strain is therefore considered
a heterologous strain in comparison to the strains contained in TIV.
Statistical analysis of data
Data from more than two groups were analyzed with a parametric ANOVA test. Tukey’s
post tests were used to compare differences among groups. Data from only two groups
were compared using the t test. Kaplan–Meier survival curves were analysed by the log rank test. Values of
*p 0.05, **p 0.01 and ***p 0.001 were considered statistically nificant. Statistical
analyses were performed with GraphPad PRISM 5.01 (GraphPad Software, La Jolla, California,
USA).