Prime-pull vaccination with a plant-derived virus-like particle influenza vaccine elicits a broad immune response and protects aged mice from death and frailty after challenge
Administered intramuscularly (IM), plant-derived, virus-like-particle (VLP) vaccines based on the influenza hemagglutinin (HA) protein elicit both humoral and cellular responses that can protect aged mice from lethal challenge. Unlike split virus vaccines, VLPs can be administered by different routes including intranasally (IN). We evaluated novel vaccine strategies such as prime-pull (IM boosted by IN) and multi-modality vaccination (IM and IN given simultaneously). We wished to determine if these approaches would provide better quality protection in old mice after less severe (borderline-lethal) challenge (ie: immunogenicity, frailty and survival).
Survival rates were similar in all vaccinated groups. Antibody responses were modest in all groups but tended to be higher in VLP groups compared to inactivated influenza vaccine (IIV) recipients. All VLP groups had higher splenocyte T cell responses than the split virus group. Lung homogenate chemokine/cytokine levels and virus loads were lower in the VLP groups compared to IIV recipients 3 days after challenge (p < 0.05 for viral load vs all VLP groups combined). The VLP-vaccinated groups also had less weight loss and recovered more rapidly than the IIV recipients. There was limited evidence of an immunologic or survival advantage with IN delivery of the VLP vaccine.
Compared to IIV, the plant-derived VLP vaccine induced a broader immune response in aged mice (cellular and humoral) using either traditional (IM/IM) or novel schedules (multi-modality, prime-pull).
KeywordsAged mouse model Frailty Influenza vaccines Virus-like particles Multi-modality Prime-pull
Cluster of differentiation
complete Roswell Park Memorial Institute medium
Cytotoxic T lymphocytes
Dulbecco’s modified eagle’s medium
Enzyme-linked immunosorbent assay
Eagle’s minimum essential medium
Fetal bovine serum
Granulocyte-macrophage colony-stimulating factor
Geometric mean titer
- H & E
Hematoxylin and eosin
Hemagglutination inhibition assay
Hank’s balanced salt solution without calcium or magnesium
Inactivated influenza vaccine
Lethal dose 50
Monocyte chemoattractant protein 1
Macrophage inflammatory protein 1 alpha
mouse lethal dose 50
Natural killer cell
- Pdm H1N1
Peridinin chlorphyll protein
Phorbol 12-myristate 13-acetate
Regulated on activation, normal T cell expressed and secreted
Roswell Park Memorial Institute medium
50% Tissue culture infective dose
Tumor necrosis factor alpha
Tissue resident memory T cells
Influenza infection can be devastating in the elderly, resulting in significant mortality and morbidity [1, 2]. In most seasonal influenza outbreaks, those ≥65 years of age typically account for 71–85% of the deaths that are relatively easy to ‘count’ [3, 4]. The impact of influenza–associated morbidity is more difficult to quantify since even a short period of forced bed-rest, either at home or in hospital, can lead to major loss of muscle mass (ie: sarcopenia)  and accumulation of other physiologic and mental deficits (ie: increased frailty) [6, 7]. More prolonged periods of bed-rest (ie: influenza complicated by pneumonia, intensive care admission) often lead to catastrophic disability with loss of independence in elderly subjects [8, 9]. Vaccination is currently the best strategy to protect the elderly from influenza viruses  but this population often responds poorly to ‘standard’ inactivated influenza vaccines (IIV) due to prior experience with influenza antigens and immunosenescence [11, 12, 13]. Although a number of vaccines that specifically target the elderly have been introduced in recent years (eg: MF59-adjuvanted IIV, high-dose (HD)-IIV) , their impact on effectiveness (ie: preventing infection) have been relatively modest [15, 16, 17, 18, 19, 20]. Their potential advantages in preventing frailty have generally not been considered.
Several highly-successful virus-like particle (VLP) vaccines are in current use (eg: HBV and HPV vaccines) and VLP vaccines have many potential advantages for a wide range of targets [21, 22, 23, 24]. Some VLP vaccines for influenza are at various stages of pre-clinical and clinical development. One of the most advanced is produced by Medicago Inc. (Quebec, QC) using transient production of the influenza hemagglutinin (HA) protein in Nicotiana benthamiana plants. After peripheral administration in mice, these plant-derived VLPs move rapidly to regional lymph nodes where they preferentially interact with B cells, NK cells and antigen-presenting cells (APC) . They also interact directly with human immune cells including B cells and APC leading to activation , internalization  and presentation . Indeed, these plant-derived VLPs appear to recapitulate many of the early interactions of intact influenza virions with host cells including fusion with host endosomal membranes . In animal models of pandemic infection, the plant-derived vaccines can provide excellent protection despite eliciting little-to-no antibody response suggesting an unusual capacity to induce cellular responses [24, 29, 30]. In clinical trials with healthy adults, the plant-derived VLP vaccines not only elicit good antibody levels against seasonal strains but also induce long-lived and poly-functional CD4+ T cell responses . The latter characteristic is of particular interest for older individuals since this population may be protected primarily by cellular immunity .
In the context of the current work, one major advantage of VLP vaccines is their flexibility: they can be administered using different routes including intramuscular (IM), intradermal (ID), oral (PO) and intranasal (IN) [32, 33]. This flexibility makes alternate vaccination strategies possible including either simultaneous or sequential administration at different sites. The former can be considered a type of multi-modality immunization that, in theory, could stimulate different, tissue-specific immune mechanisms. The latter approach, sometimes referred to as ‘prime-pull’, consists of a systemic “priming” dose (eg: IM) followed by a local “pull” dose given at the site of natural infection to ‘recruit’ antigen-specific immune cells to that area (eg: PO or IN) [34, 35, 36]. These alternate vaccination strategies could potentially provide better protection in the elderly by inducing a long-lasting, cross-protective cellular response [37, 38, 39] and boosting of local mucosal immunity [34, 40]. As noted above ‘standard’ vaccination strategies based on IM delivery of IIVs that primarily elicit systemic antibodies have had only limited success in the elderly [31, 41]. We were interested to know if the flexibility and unusual immunogenicity of the plant-derived VLP vaccines could be exploited to better protect older individuals. We have recently shown that a single dose of a plant-derived H1-VLP candidate vaccine can protect old mice from a lethal A/California/07/2009 H1N1 challenge . To our surprise, a single dose of the same VLP vaccine administered IN protected ~ 60% of the animals despite the complete absence of a detectable systemic serologic response .
In the current work, we extended these observations by testing alternate VLP immunization strategies and following immunogenicity as well as protection against both frailty and death following a borderline-lethal A/California/07/2009 H1N1 challenge. Our results confirmed that the VLP vaccine elicits a broader immune response than IIV regardless of the vaccination strategy used. Animals that received a dose of the VLP vaccine IN had the most rapid weight recovery and the least change in frailty index after challenge infection. Although preliminary, these data suggest that such alternate vaccination strategies should at least be considered for elderly subjects when vaccines with the flexibility to be administered via multiple routes become commercially available.
Infection survival rates
Cellular immune response
CD4+ T cells
CD8+ T cells
Antigen-specific CD8+ T cell cytokine responses were more variable than CD4+ responses but were still more consistently observed in the VLP-vaccinated animals than antibody responses. Overall, poly-functional CD8+ T cell responses above mean PBS levels were found in 40% of the VLP-vaccinated animals and 0% of the IIV-vaccinated mice (Additional file 1: Table S3). Again, the VLP-IM/IN and VLP-IM/IM groups had the most convincing CD8+ T cell responses; generally, for the VLP-IM/IN groups (poly-functional and all individual cytokines) and for IL-2 and IFNγ in the VLP-IM/IM group (reaching significance for IFNγ versus PBS: p < 0.05) (Fig. 4b). Again, the IIV-IM/IM group mounted little-to-no CD8+ T cell response above baseline levels except for IL-2 production (0.03% versus 0.001% in the PBS group) although this difference did not reach statistical significance.
Lung viral loads, histology and cytokine/chemokine concentrations 3 dpi
Summary of total histopathology scores (20 points total) from H&E stain at 3 days post-infection
7.83 ± 2.25
13.25 ± 0.75
5.25 ± 3.27
IM + IN
6.00 ± 3.29
6.38 ± 3.58
Two of the groups that that received VLP vaccines (VLP-IM/IM and VLP-IM/IN) lost the least amount of weight (− 9.4 ± 1.5% and − 9.9 ± 1.5% respectively) and recovered most rapidly, returning to near baseline weights by 18 dpi (Fig. 5b). The PBS control lost the most weight (− 16.1%) and remained well below their baseline weight at 18 dpi (− 6.8 ± 2.0%). The VLP-IM + IN and IIV-IM/IM groups were intermediate in both their maximum weight loss (− 13.3 ± 1.9% and − 12.7 ± 1.4% respectively) and the timing of weight recovery (still − 3.1 ± 2.4% and − 1.5 ± 1.1% at 18 dpi respectively).
The development of better influenza vaccines for the elderly is not only a major problem; it is also a rapidly growing problem as most of industrialized countries of the world continue their epidemiologic transition towards the ‘older end’ of the age spectrum . Immune responses to influenza vaccination in older subjects are subject to a wide range of influences including a life-time of exposures to wild-type viruses as well as vaccines [13, 44] and loss of immune competence due to thymic involution, CMV infection, chronic inflammation and other factors (ie: immunosenescence and/or inflamm-aging) [45, 46, 47, 48]. It should therefore be no surprise when vaccines that work reasonably well in children and healthy young adults fail to work in the vulnerable elderly population. This is particularly true since a great deal of effort has been expended to optimize the ability of standard IIVs to elicit antibodies, and specifically antibodies detected by the classical hemagglutination inhibition (HI) assay [41, 49], when it seems increasingly clear that the elderly are protected primarily by other immune mechanisms including T cell responses .
New vaccines and vaccination strategies that elicit a different pattern of immunity from that induced by IIVs are clearly needed to provide better protection in older individuals. Among the many novel influenza vaccine candidates moving forward through pre-clinical and clinical testing , the plant-derived VLP vaccines that were the focus of the current work have many attractive features. They have considerable flexibility regarding route of administration [42, 51], they are efficiently delivered to lymph nodes and APCs [25, 26, 27, 28] and they have been shown to elicit both humoral and cellular responses in animal models and human trials [23, 24, 30, 42, 51]. In addition to new vaccines and strategies, it is also important to use appropriate animal models in developing better influenza vaccines for the elderly . Although ferrets are widely viewed as the best animal model for influenza infection , they live for much longer and are much more expensive than mice, not to mention the limited availability of immunologic reagents  and their very large teeth. Indeed, we are aware of only a single study of influenza vaccination or infection conducted in aged ferrets . In contrast, aged mice have been used in influenza and influenza vaccine research for at least 40 years due to their relatively short life-spans, their immunologic tractability and their relatively low cost. However, their limitations as models for human elderly should be acknowledged. For example, even very old mice are typically influenza naïve when used in studies rather than having had a life-time of varied exposures to different influenza strains and vaccines . Of course, as with any complex human disease, immune responses in mice are not always fully predictive of response in humans (‘mice lie, monkeys exaggerate’) . Nonetheless, the loss of antibody response despite strong antigen-specific cellular reactivity that we observed in our aged animals following VLP vaccination (Figs. 3 and 4) is certainly consistent with Medicago’s on-going studies of the Quadrivalent influenza VLP vaccine in older subjects (unpublished data). Knowing what to study in aged mice is also critically important. As recently pointed out by Miller et al., we must first ‘know ourselves’ (ie: have a better understanding of the immune correlates of protection) in order to know what kind of immune response we want following vaccination . Continuing this line of reasoning for a moment, it is also important that we know what outcomes to assess. Historically, almost all influenza vaccination studies in aged mice have focused on immunologic parameters (usually just antibody responses as discussed above) and survival. It is very likely that this relatively narrow focus has hampered the development of novel vaccines and vaccination strategies. The recent description of protocols to assess frailty in aged rodent models  has been a major advance for influenza vaccine studies. Although complex and time-consuming, the inclusion of frailty assessments in our current work permitted us to recognize subtle differences between vaccines and vaccination schedules that may be highly relevant to protecting the elderly population.
Although some aspects of our work are best considered preliminary, our overall results supported the greater immunogenicity of the VLP vaccine compared to IIV and raised the possibility that delivery of the VLP vaccine IN might have advantages in terms of pattern of immunity generated, lung inflammation and frailty. Although weight loss and mortality among the animals that received any VLP regimen were highest in the VLP-IM + IN group, several other outcomes were very similar between the VLP IM/IN and VLP IM + IN groups including viral loads and histopathology. Lung homogenate cytokine/chemokine profiles were strikingly different in the VLP-IM + IN and VLP-IM/IN animals however (Fig. 6). Despite considerable mouse-to-mouse variability, the VLP-IM + IN animals had much higher levels of pro-inflammatory chemokines/cytokines (IL-Iβ, IL-6, MIP1-α, IFNγ) than the VLP-IM/IN group 3 dpi: a pattern much closer to that seen in the IIV-IM/IM group. Since very little is known about IN dosing of VLP vaccines, a small follow-up study was performed to assess a higher dose of VLPs using the VLP-IM + IN schedule (3 μg/route at each time-point instead of 1.5 μg). Summary data comparing these two doses (Additional file 3: Figure S2) strongly suggest that the 1.5 μg dose used in this study was sub-optimal for IN delivery.
Overall, the conventional IIV-IM/IM strategy was inferior to one or both 3 μg VLP strategies (VLP-IM/IM and VLP-IM/IN) for all outcomes except survival. It is interesting that increasing the dose in the VLP-IM + IN strategy to 3 μg/route not only changed the cytokine/chemokine profile and significantly reduced viral titres at 3 dpi (p < 0.01), it also increased survival (84.2%) and dramatically increased lung-resident T cell responses (Additional file 3: Figure S2). Although lung-resident T cells were barely detectable in other groups, the VLP-IM + IN (6 μg) animals had a significantly higher number of lung tissue-resident memory (TRM), antigen-specific CD4+ T cells (p < 0.01) and a higher proportion (%) of antigen-specific CD8+ T cells expressing IFNγ (Additional file 3: Figure S2). TRM CD8+ T cells may be critical in viral clearance [38, 60, 61] and TRM CD4+ cells are also thought to be important for influenza protection [62, 63]. Indeed, TRM CD4+ cells appear to be a good marker for protection from morbidity and mortality in murine models of influenza infection [39, 64]. Moreover, CD4+ T cell epitopes are relatively conserved across different influenza strains [65, 66] and TRM CD4+ cells may play a particularly important role in protecting against reinfection [39, 64]. Pre-existing, antigen-specific, peripheral blood CD4+ T cells have also been shown to be a good correlate of immunity in human challenge studies . It is interesting that an early trial with a live-attenuated H3N2 vaccine by Treanor and colleagues demonstrated better protection in elderly subjects who received simultaneous IIV + attenuated virus IN compared to IIV alone . Although a small number of investigators have pursued prime-pull strategies for seasonal vaccination in children  or pandemic vaccines in adults [69, 70], the number of vaccine options has been limited and there have been no further studies in the elderly to our knowledge. Together, these observations and our data strongly suggest that multi-modality or prime-pull strategies may have important advantages for elderly subjects.
In conclusion, we exploited the flexibility of the VLP vaccine and our aged mouse model to compare the standard vaccine (IIV-IM/IM) and the novel vaccine delivered IM (VLP-IM/IM) as well as multi-modality (VLP-IM + IN) and prime-pull (VLP-IM/IN) strategies. Each of these approaches was assessed using both conventional methods (eg: viral loads, survival curves, classic serologies) as well as less common methods including splenocyte and tissue-resident memory T cell responses, lung cytokine/chemokine profiles and frailty to assess the potential for novel approaches to improve vaccine-induced protection in the elderly. Our findings strongly support the further exploration of such alternative vaccination strategies in older subjects.
Virus, mice and vaccines
Female BALB/c mice (18–22 months of age: Charles River Laboratories, Montreal, QC) were vaccinated twice on day 0 (d0) and day 21 (d21). All active vaccinations were based on hemagglutinin (HA) content for H1N1 A/California/07/2009 (pdmH1N1). The plant-derived H1-VLP vaccine was produced by Medicago Inc. (Quebec City, QC) as previously described  using the wild-type HA sequence from pdmH1N1. Three groups of animals received the VLP vaccine: i) two 3 μg doses intramuscularly (IM/IM) ii) a first 3 μg dose IM boosted at d21 by a 3 μg dose intranasally (IM/IN: Prime-Pull group) or iii) two doses of 1.5 μg IM plus 1.5 μg IN (IM + IN: Multi-modality group). The active comparator group received two 3 μg doses of a split inactivated influenza vaccine (H1N1 A/California/07/2009) (IIV: BEI resources, Manassas, VA). Control animals received similar volume IM and/or IN ‘mock’ vaccinations with phosphate buffered saline (PBS: pH:7.4, Wisent, Saint-Bruno, QC). For H1-VLP and PBS injections IM, 50 μL was administered into the right quadriceps muscle using a 28G½ needle. For IIV injections, 50 μL was injected into each quadriceps muscle (100 μL total). Instillations of H1-VLP or PBS IN (25 μL/nare) were performed in lightly isoflurane anesthetized mice (50 μL total).
Peripheral blood was collected using microtainer serum separator tubes (BD Biosciences, Mississauga, ON) from the lateral saphenous vein at d0 (pre-vaccination) and at d21 (data not shown) and d42 after the first dose of vaccine (Fig. 1). Serum was stored at − 20 °C in aliquots until used. At d42 (immediately pre-challenge), approximately 1/2 of the animals were sacrificed within isoflurane and a CO2 chamber (typically 6–8 mice/group). A terminal serum sample was collected by cardiac puncture. Spleens and lungs were harvested from individual animals and processed as described below. The remaining mice (typically 6–8 mice/group) were scored for frailty on d40–42 then challenged with a sub-lethal dose of wild-type H1N1 A/California/07/2009 virus (525 TCID50 in 50 μL: National Microbiology Laboratory, Public Health Agency of Canada) by IN instillation (25 μL/nare). Weight loss was monitored daily for up to 28 days. At d45 or 3 days post-infection (dpi), 3–5 mice/group were sacrificed (isoflurane/CO2) and serum (cardiac puncture) and lungs were collected (viral load and cytokine analysis). At d67 (25 ± 4 dpi), surviving mice were scored for frailty and sacrificed as above to collect serum and lungs.
Antibody titre measurements
Serum antibody levels were measured by hemagglutination inhibition assay (HAI), microneutralization assay (MN) and enzyme-linked immunosorbent assay (ELISA) as previously described .
Lung T cell isolation and stimulation
Lungs were perfused with 10 mL of PBS and collected in in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS (both Wisent, St. Bruno, QC) stored on ice for approximately 2 h before transferring to a lung digestion cocktail (DNAse I (10 mg/mL: Sigma, St. Louis, MO), Collagenase (12.5 mg/mL, Sigma), Liberase (10 mg/mL: Roche, Basel, Switzerland), hyaluronidase I (50 mg/mL, Sigma)) prepared in DMEM. After a 30–40 min incubation at 37 °C in 5% CO2, lungs were processed individually through a 70 μm cell strainer, resuspended in 10 mL of Hanks Buffered Salt Solution without calcium/magnesium (HBSS −/−: Wisent) then centrifuged at 320×g for 8 min at 4 °C. Cells were passed through a cell strainer for a second time and washed with 5 mL of HBSS−/− before re-suspension and counting in complete Roswell Park Memorial Institute medium (cRPMI). Lung cells were seeded in duplicate at 1.5 × 106 cells/well in 96-well U-bottom plates (BD Falcon, Mississauga, ON) in 200 μL. Cells were stimulated with cRPMI alone (unstimulated), PMA+ ionomycin (each 1 mg/mL: Sigma) or with 4 μg/mL of a previously-described  overlapping H1 peptide pool, from pdmH1N1 (BEI resources, Manassas, VA) (all stimuli at 80 μL/well). Plates were incubated for 5 h at 37 °C in 5% CO2.
Splenocyte isolation and stimulation
Individual spleens were harvested at d42 into HBSS −/− (Wisent) and processed at room temperature (RT) as previously described . Isolated splenocytes were seeded in duplicate in U-bottom 96-well plates (1 × 106 cells/well) as above and stimulated with cRPMI or alone (unstimulated), H1-VLP (2.5 μg/mL HA) or PMA+ ionomycin (each 1 mg/mL: Sigma) for 18 h at 37°Cat 5% CO2 (all stimuli at 100 μL/well).
T cell responses was assessed in mononuclear cells isolated from the lungs and spleen at d42 (immediately pre-challenge). Golgi Plug™ (BD Science, San Jose, CA) and added to lung cells at the beginning of stimulation or 13 h after stimulation for splenocytes (20 μL /well). For flow cytometry, cells were washed twice with cold PBS then centrifuged at 320×g, 8 mins at 4 °C. Viability dye (50 μL/well) (Affymetrix ebioscience, Waltham, MA) was added to each well (1:10 for lung cells and 1:100 for splenocytes in PBS) and incubated for 20 min at 4 °C. Cells were washed as above and Fc block (1 μL/well, BD Science, San Jose, CA) was added. The following cocktail was used for surface stains: CD3 –FITC (Clone: 145-2C11, eBioscience), CD4-V500 (Clone: RM4–5, BD Bioscience) and CD8-PerCP-Cy5 (Clone: 53–6.7, BD Bioscience), CD45-BUV495 (Clone: 30-F11, ebioscience), CD11a-APC (Clone: M17/4, Biolegend, San Diego, CA), CD103-BV711 (Clone:M290, BD Bioscience) and CD69-BV605 (Clone:H1.2F3, Biolegend). After 30 min, cells were washed as above, then fixed overnight at 4 °C with 100 μL of fixative (BD Science). For the intracellular stains, cells were washed as above except with 1X permeabilization buffer (BD Science), then stained with an intracellular cocktail containing: IL-2-Pe-Cy5 (Clone: JES6-5H4, Biolegend), IFNγ-PE (Clone: XMG1.2, BD Science) and TNFα-efluor450 (Clone: MP6-XT22, Affymetrix ebioscience) and incubated for 40 min in the dark at 4 °C. After washing with PBS as above, cells with fixed with an intracellular fixative (Affymetrix ebioscience) and analyzed on BD LSRFortessa X-20 (BD Science) using Flowjo software (version 10.0.8r1). Our gating strategy is shown in Additional file 2: Figure S1.
Frailty measurements were adapted from Whitehead et al  using 29 of the original 31 parameters to adapt the procedure to BALB/c mice. The parameters assessed fell into the following categories: integument, musculoskeletal, ocular and nasal, vestibulocochlear/auditory, digestive, urogenital, respiratory. Signs of discomfort, body weight and temperature were also assessed. Additional file 1: Table S1 is the scoring sheet that was used at d0, d42 and d67 (25 dpi). Deficits were measured using a 3-point scale: 0 = no deficit, 0.5 = mild deficit and 1 = severe deficit. All measurements were performed by the same operators (BH or AB) who were blinded to group assignment. To correct for a survivor effect, animals that died were assigned the highest Frailty Index score of a surviving animal in any group.
Lung viral load and cytokine/chemokine levels at day 45 (3 dpi)
Both lungs were collected at 3 dpi and homogenized for viral load and cytokine/chemokine measurements as previously described [42, 51, 74]. Briefly, viral titres were calculated from the supernatants of lung homogenates using the Karber method and reported as log10 50% tissue culture infectious dose (TCID50): logTCID50/0.1 mL = − 1 - (observed lysis of monolayer (as a percent(%) /100–0.5) x log10 . Viral load data are representative of 3–5 mice/group from two independent experiments. The lung homogenate supernatants were used (1:5 and 1:10) to measure 16 tissue cytokine/chemokine concentrations using a multiplex ELISA (Quansys, Logan, UT). Lung homogenates were collected from 4 to 7 mice/group in one experiment and tested in duplicate.
Lung samples from one lung that was excised, fixed and processed for H&E staining as previously described . Briefly, lung samples were fixed in 10% formalin (Fisher Scientific, Ottawa, ON) then embedded in paraffin (Leica, Concord, ON). Sections (4 μm) were applied to slides with a cover slip and scored at 10X and 100X magnification. Slides were scored by a blinded operator (BJW) using a 5 parameter scoring protocol 1) airway epithelial necrosis, attenuation or disruption, 2) airway inflammation, 3) peribronchiolar & perivascular lymphocytic cuffing, 4) alveolar cellular exudate/edema and interlobular edema and 5) alveolar septal inflammatory cells and cellularity . Each parameter was scored from 0 to 4 for a total possible score of 20.
The geometric mean ratios between groups and their 95% confidence intervals (CI) were calculated. For statistical analysis, one-way ANOVA was performed on HAI, ELISA, MNs, viral titres and frailty scores. For survival statistics, a log-rank (Mantel-Cox) test was used. All other statistical analyses used two-way ANOVA. All analyses were performed using GraphPad Prism 6.0 software.
We would like to thank Annie Beauchamp, Kaitlin Winter, Janna Shapiro, Angela Brewer and Louis Cyr for animal and technical assistance. We thank the immunophenotyping Core of the Research Institute of the McGill University Health Centre.
The experiments were designed by BH, SP, BJW and NL. BH performed all experiments. All authors contributed in the preparation of the final manuscript. All authors read and approved the final manuscript.
This work was supported in part by a Canadian Institutes of Health Research grant to BJW (#34469) and an academic-industry team award led by BJW that was co-funded by Medicago Inc. and the Ministère de l’Économie et de l’Innovation du Québec with project oversight by Genome Quebec. The company also provided research materials. BH held a studentship from the Research Institute of the McGill University Health Centre.
Ethics approval and consent to participate
All procedures were carried out in accordance with guidelines of the Canadian Council on Animal Care, as approved by the Animal Care Committee of McGill University.
Consent for publication
BJW has served as the medical officer for Medicago Inc. since 2011 and has held peer-reviewed grants with the company from various sources. SP and NL are currently both employees at Medicago Inc.
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