1. Introduction
By impacting the magnitude, duration, and nature, of the immune response, adjuvant selection can make, or break, a vaccine candidate [
1]. Despite this, greater priority is often placed on antigens while relying on historic, empirical approaches to adjuvant selection and optimization. More recently, there has been priority to expand the panel of adjuvants available for human and livestock vaccines and to develop a greater understanding of their mechanisms of action (MOA) [
2].
Many consequences of adjuvant administration have been described, ranging from localized depot effects [
3], enhanced uptake and presentation of antigens [
4,
5], to higher-order impacts on immune response, including modulation of cytokine and chemokine profiles [
6,
7,
8] and recruitment of immune cells [
8,
9]. Increasingly, there are efforts to elucidate different molecular MOA of adjuvants through omics approaches within the context of systems vaccinology [
2]. Here the priority is to gain insight into the cellular responses induced by different adjuvants, at different biological locations, and to correlate these induced responses with various indicators of vaccine efficacy. To this objective, transcriptional responses has frequently been applied to define responses to common adjuvants [
10,
11,
12,
13,
14]. From these efforts, a panel of “adjuvant core response genes” have been identified, as well as defining various molecular effectors including cytokines, chemokines, innate immune receptors, interferon-induced genes, and adhesion molecules [
6]. While transcriptional profiling offers some advantages in terms of the maturity of the technology, ease of application, and magnitude of coverage, the findings must be interpreted with the caveat of potential disconnect between transcriptional and phenotypic responses due to post-transcriptional and post-translational regulatory events.
Kinase-mediated protein phosphorylation is the central mechanism for regulation of many cellular processes, including those associated with innate and adaptive immunity [
15]. Investigations of global patterns of kinase-mediated signaling (kinome analysis) has emerged as a powerful tool to understand complex cellular biology [
16]. Within that, kinome peptide arrays have demonstrated utility for deciphering immunity, including characterizing host-pathogen interactions [
17,
18,
19], anticipating individual responses to vaccination [
20], and describing cellular responses to discrete molecules (such as pathogen-associated molecular patterns) that are often employed as vaccine adjuvants [
21,
22]. Tissue and individual-specific signaling differences have been defined [
23], supporting the use of the technology to define individualized responses to vaccines as well as characterizing different routes of vaccine delivery and unique sites of adjuvant action. The technology has proven robust in defining responses in biologically complex samples, including peripheral blood mononuclear cells [
20], muscle and gut tissue [
19,
24], and whole insect homogenates [
25]. Collectively, kinome analysis appears a strong candidate to be included within systems vaccinology approaches to define adjuvant MOA.
Polyphosphazenes are a novel category of adjuvants shown to be effective in a range of species through both parenteral and mucosal delivery [
26,
27]. These high molecular weight synthetic polymers consist of a backbone of alternating phosphorus and nitrogen atoms with organic side groups anchored to the phosphorus atoms [
28,
29]. Of the polphosphazenes, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), has demonstrated considerable ability to promote high-titre, long-lasting, immune responses to several antigens [
30,
31,
32,
33]. Through systemic and mucosal administration, including respiratory, oral, rectal, and intravaginal routes, PCEP promotes a balanced Th1/Th2 type response [
26,
27], and functions well in combination with other adjuvants [
34]. These characteristics prompted interest to advance PCEP as an adjuvant for human and veterinary applications.
There is growing appreciation of the biological complexity of the MOA of polphosphazenes. Indications of their immunostimulatory properties indicate that the formation of depots at the injection site offer minimal contribution to their adjuvant activity [
31], although complexes with the antigen do facilitate antigen delivery to immune cells [
35]. Recent efforts attribute their activity to stimulation of immune responses at the injection site [
4], splenocytes [
27,
36], and lymph nodes [
37]. The potential of PCEP as an adjuvant for human and animal vaccines, coupled with indications of a diverse and biological complexity MOA, make it an ideal candidate for investigation of kinome analysis to describe molecular responses to adjuvants.
2. Materials and Methods
2.1. Adjuvants
PCEP was generated by Idaho National Laboratory (Idaho Falls, ID, USA) using described methods [
36,
37]. Endotoxin levels within PCEP were determined to be less than 0.034 ng/mL as assessed by the Limulus Amebocyte Lysate assay (Biowhittaker, Walkersville, MD, USA). PCEP was dissolved in Dulbecco’s phosphate buffered saline (PBS) (Gibco, Grand Island, NY, USA). The alum adjuvant (Thermo Fisher Scientific, Rockford, IL, USA) is a mixture of alum and magnesium hydroxide (40 mg/mL).
2.2. Mouse Trials
Female BALB/c mice (purchased from Charles River Laboratories, North Franklin, CT, USA) of 4–6 weeks of age were used in these experiments. The animal experiments were approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane use of animals.
2.3. Quantitative Real-Time PCR (qRT-PCR)
Female BALB/c mice (
n = 6) were injected intramuscularly (i.m) in the quadriceps muscle with 25 μL of either phosphate-buffered saline (PBS) as control or 50 μg PCEP per animal. As the trauma caused by injecting a liquid into the tissue is sufficient to alter gene expression [
6,
38], the PBS-injected group is an important control. Mice were euthanized and samples collected at 24 h post-injection.
Immediately after mice were euthanized, whole muscle tissues from the thigh were collected in TRIzol (Invitrogen) and aseptically homogenized with 2.3 mm Zirconia microbeads (Biospec Products Inc., Bartlesville, OK, USA) in a Mini-Beadbeater
TM (Biospec Products Inc., Bartlesville, OK, USA). The homogenates were centrifuged for 1 min at 10,000×
g, and the supernatants were collected for total RNA extraction as per the manufacturer’s instruction. The extracted RNA was quantified and treated with DNase (Invitrogen). The cDNA was synthesized using random hexamers (Applied Biosystems) and SuperScript
®® II Reverse Transcriptase (Invitrogen) as per manufacturer’s instruction. All PCR reactions were carried out in duplicate in 96-well plates with optical quality tape (Bio-Rad) using an iCycler iQ
®® Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each PCR reaction contained 1 μL target cDNA, 0.2 μM each of forward and reverse primers, 7.5 μL of iQ SYBR
®® Green Supermix (Invitrogen) and distilled water to 15 μL of final volume according to manufacturer’s instruction. The negative control contained all the reagents except cDNA. All the primers used in quantitative RT-PCR are shown in
Table 1. Reference genes GAPDH, RPL19 and18 s rRNA were analyzed and the best (GAPDH) was selected for further analysis. Amplification was performed by initial denaturation at 95 °C for 3 min in cycle 1, followed by cycle 2 (95 °C, 15 s; 55 °C, 30 s; 72 °C, 30 s) ×45 and then cycle 3, the Melt curve analysis, was pre-set at 55 °C ramping to 95 °C with 1 °C increase each 10 s and final hold at 20 °C. A Melt Curve analysis was performed to ensure that any product detected was specific to the desired amplicon.
2.4. Isolation of Recruited Cells from Site of Injection and Draining Lymph Node
Female BALB/c mice were divided into three groups (n = 5 per group) and were injected i.m on both legs with 25 μL of either PBS as control, 50 μg PCEP, or 0.5 mg of alum. At 24 h post-injection, muscle tissues were dissected from the site of injection, minced, and incubated with digestion buffer (Hank’s Balanced Salt Solution [HBSS] [Gibco] supplemented with 0.1% type II collagenase D [Worthington Biochemical, NJ, USA], 0.2% BSA [Sigma–Aldrich, MO, USA], 0.025% trypsin [Gibco] and 0.01% DNase I [Roche Diagnostics, Germany]) for 45 min at 37 °C under constant agitation. The cell suspension was filtered through 70 μm cell strainer and layered on 25% percoll (GE healthcare, Chicago, IL, USA) and centrifuged at 2000× g for 1 h. The cell pellets were washed twice, resuspended in RPMI (Gibco) with 10% FBS (Gibco), and used for fluorescent labeling for FACS analysis. Cell viability was estimated by Trypan Blue (Gibco) exclusion.
The draining inguinal lymph nodes were dissected, minced, and incubated with digestion buffer containing 2 mg/mL collagenase D (Roche Diagnostics, Mannheim, Germany) and 0.25 mg/mL DNase I in HEPES (Gibco) for 15 min at 37 °C. These samples were then filtered through 70 μm cell strainer to obtain a single cell suspension which was used for fluorescent labeling for FACS analysis.
2.5. Flow Cytometry
For FACS analysis, all the cells were stained in the presence of an Fc block. For staining, cells were incubated for 20 min at 4 °C using the following antibodies: CD11b-FITC, Ly6C-APC, Ly6G-APC, F4/80- PE, CD11c-PE, CD3-APC, CD8-FITC, CD4-FITC, CD19-FITC (all from eBiosciences, CA, USA) and CD8-PerCP-Cy5.5, CD4-PerCP-Cy5.5 (all from BD Biosciences). The following markers were used to identify specific cell types: monocytes (CD11b+ Ly6C+), neutrophils (CD11b+ Ly6G+), macrophages (F4/80), dendritic cells (CD11c), B cells (CD19), CD4+ T cells (CD3+ CD4+) and CD8+ T cells (CD3+ CD8+).
Unstained cells were used to set up the instrument. Compensation controls were set up using single stains and isotype controls were used to determine the level of non-specific binding. The cells were gated based on simple forward and side scatter patterns. Furthermore, all the dead cells were excluded using the viability dye, propidium iodide and doublet discrimination was performed by plotting FSC-H vs. FSC-A. If we take the example of lymphocytes, they were identified and gated by their forward and side scatter patterns. The CD3+ T cells were then further identified and gated by the expression of CD4+ and CD8+. The expression of surface markers was assessed using CellQuest analysis software on a FACSCalibure flow cytometer (BD Biosciences).
2.6. Peptide Arrays for Kinome Analysis
Female BALB/c mice were divided into three groups (n = 5) and were injected i.m on both legs with 25 μL of either PBS as control, 50 μg PCEP, or 0.5 mg of alum. At 24 h post injection, muscle tissues were dissected from the site of injection, minced, and incubated with digestion buffer (Hank’s Balanced Salt Solution [HBSS] [Gibco] supplemented with 0.1% type II collagenase D [Worthington Biochemical, Lakewood, NJ, USA], 0.2% BSA [Sigma–Aldrich, MO, USA], 0.025% trypsin [Gibco] and 0.01% DNase I [Roche Diagnostics, Germany]) for 45 min at 37 °C under constant agitation. The cell suspension was filtered through 70 μm cell strainer and layered on 25% percoll (GE healthcare, Chicago, IL, USA) and centrifuged at 2000× g for 1 h. The cell pellets were washed thrice with ice-cold PBS and stored at −20 °C until further use.
Similarly, the draining inguinal lymph nodes were dissected, collected, minced, and incubated with digestion buffer containing 2 mg/mL collagenase D (Roche Diagnostics, Mannheim, Germany) and 0.25 mg/mL DNase I in HEPES (Gibco) for 15 min at 37 °C. It was then filtered through 70 μm cell strainer to obtain a single cell suspension. The cells were washed thrice with ice-cold PBS and stored at −20 °C until further use.
Protocols for the design and application of the peptide arrays have been described [
20]. Arrays were constructed by a commercial provider (JPT Innovative Peptide Solutions, Berlin, Germany and designed to include peptides representing phosphorylation events associated with a wide variety of signaling pathways. Each array includes nine technical replicates of each of the 282 unique peptides. All kinome experiments were performed on the same day to minimize potential inter-assay variance.
2.7. Analysis of Kinome Data
Peptide-spot intensities were transformed using a variance-stabilizing normalization (VSN) method through the online software, PIIKA 2.0 (
https://saphire.usask.ca/saphire/piika2.0/) [
39]. Peptides that showed variation in technical replicates via Chi-squared test (χ
2 < 0.01) were removed from subsequent analysis. Consistent technical replicates were averaged together, and fold-change (FC) for each peptide was calculated as previously described [
20]. Theta-distributed stochastic neighbour embedding(t-SNE) analysis and hierarchical clustering were conducted using peptides with consistent phosphorylation (χ
2 > 0.01). The t-SNE analysis was conducted using the R package Rtsne (
https://github.com/jkrijthe/Rtsne) (accessed on 14 June 2021) and visualized using ggplot2 (
https://ggplot2.tidyverse.org) (accessed on 14 June 2021). The t-SNE analysis was performed 100 times and the result with the lowest value of the objective function was selected. The construction of the heatmap using PIIKA 2.0 has been previously [
39]. Hierarchical clustering was conducted using the Pearson correlation distance and McQuitty linkage. Peptides were considered differentially phosphorylated under two given criteria: first, the peptide was consistently phosphorylated according to the Chi-squared test and second, the VSN-transformed phosphorylation intensity of an individual peptide was significantly different (two-tailed Welch’s t-test for Unequal Variances,
p < 0.05) between cohorts.
2.8. Pathway Over-Representation Analysis
Peptides that were differentially phosphorylated were subjected to pathway over-representation analysis (ORA) using InnateDB [
40]. ORA was completed using the hypergeometric algorithm with Benjamani–Hochberg correction method, and pathways were considered statistically significant with a false discovery rate (FDR) of
p < 0.05.
2.9. Statistical Analysis
The increase in target gene expression levels in PCEP stimulated muscle tissues were calculated as fold change increase (2−ΔΔCT). The data for cell recruitment were analyzed using Graph-Pad Prism 6 software (GraphPad Software, San Diego, CA, USA). Differences in the cell numbers between the treatments were analyzed by two-way ANOVA by Ranks and the significant differences between the treatments were compared by Bonferroni multiple-comparison test where **** p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05.
4. Discussion
Within this investigation, kinome profiling was performed to define signaling events to PCEP at the injection site and draining lymph node at twenty-four hours post-injection. At the injection site, PCEP induced pro-inflammatory signaling, as exemplified by the Jak-Stat pathway. PCEP-induced activation of interferon-based signaling was supported by elevated expression of proinflammatory genes as well as recruitment of immune cells. This supports the hypothesis that an element of the MOA of PCEP resides in its ability to promote a pro-inflammatory environment at the injection site. Kinome analysis also indicates that responses to PCEP are not restricted to the injection site as strong signaling responses, relative to alum and distinct from those observed at the injection site, occur at the draining lymph node. Consistent with that, PCEP resulted in significantly higher recruitment of immune cells to the lymph node than alum. Collectively, these results indicate that the complex, multi-faceted adjuvant activity of PCEP and support the utility of kinome analysis to define adjuvant MOA.
In 2015, Hagan and Fox predicted a “New Golden Age” for vaccine adjuvants [
42]. This enthusiastic assessment was based on an expanding knowledge of adjuvant MOA that was largely enabled through systems vaccinology approaches. While initially overlooked, we now have fuller appreciation of the ability of adjuvants to improve the range, practicality, and efficacy of vaccines through dose sparing, enabling rapid immune responses, broadening of the induced antibody response, and optimizing of the magnitude of the vaccine-associated antibody response [
1,
2]. Adjuvants can also be the critical determinants of new categories of vaccines, including for the induction of T cell responses, mucosal vaccines, and personalized vaccines. This includes interest in individual adjuvants with these characteristics as different formulations and co-formulations. Within this power to impact immune responses, there is also the appreciation of the potential for unintended, and potentially detrimental responses, both at the site of administration as well as systemic consequences [
43].
The value of systems vaccinology is to identify molecular responses to immunization to identify surrogate markers of immunogenicity and reactogenicity that anticipate whether the patient will develop the desired immune response (correlates of immunity) and/or will be protected from the targeted disease (correlates of protection), as well as to understanding the underlying mechanisms of these outcomes [
44]. These responses occur at the site of injection as well as other immune related locations, such as peripheral blood mononuclear cells and lymph nodes. While systems vaccinology approaches have largely been grounded in transcriptional analysis, that various adjuvants are known to activate signaling pathways associated with innate and adaptative immune responses, coupled with recent advances in technologies for defining global patterns of phosphorylation-mediated signal transduction, there is both opportunity and priority to apply kinome analysis to define adjuvant MOA. The motivations for the current investigation were to define biological responses to an important adjuvant, including considerations of localized and systemic effects, as well as to investigate kinome analysis as a tool to define adjuvant MOA.
With respect to the MOA of PCEP, the current work supports the hypothesis this adjuvant functions through both localized and systemic immune responses. At the site of injection, there is clear indication of activation of proinflammatory signaling beyond that resulting from general tissue damage [
38]. This offers mechanistic explanation and additional dimension of the observed patterns of induced expression of a variety of proinflammatory genes. There have also been indications that the MOA of PCEP is not limited to the site of injection with indication of activation of higher-order immune response as suggested by patterns of increased patterns migration of immune cells to lymph nodes in response to PCEP administration [
37]. With that, however, there was minimal information about the biochemical basis of these changes, including potential insights into biomarkers, as well as comparable analysis of these changes to other adjuvants. The kinome analysis of the responses within the lymph nodes to PBS, alum, and PCEP provides context of the magnitude of systemic responses induced by PCEP relative to alum; a difference of 100 versus 7 differentially phosphorylated peptides. Within those signaling responses, there are the anticipated changes to signaling associated with inflammatory responses as well as activation of erythropoietin (EPO) mediated signaling. Within mouse models, it has been demonstrated that administration, or engineered overexpression, of EPO increased humoral antibody responses to several antigens [
45]. Similarly, administration of EPO to patients with chronic kidney disease improved vaccine responsiveness [
46]. Erythropoietin treatment is also associated with an augmented immune response to the influenza vaccine in hematologic patients [
47]. Collectively this suggests a role for EPO in humoral immune responses. The implication that PCEP administration influences EPO signaling within the lymph nodes merits further investigation as a potential MOA. Collectively, the results of this investigation highlight and detail the complex, multi-faceted MOA of PCEP while supporting the utility of kinome analysis as a tool to define responses to adjuvants.