Edited by: Urszula Krzych, Walter Reed Army Institute of Research, United States
Reviewed by: Arun Kumar, Health Sciences North, Canada; Raffael Nachbagauer, Icahn School of Medicine at Mount Sinai, United States; Christophe Chevalier, Institut National de la Recherche Agronomique (INRA), France
†These authors have contributed equally to this work.
Specialty section: This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology
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Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis.
DNA vaccines were introduced over two decades ago. This very promising technique relies on the production of an antigen by the cells of an immunized host after the introduction of a genetically engineered expression cassette for this antigen and, as a consequence, induction of humoral and cellular immune responses (
The most promising approaches involve the use of NF-κB (
Influenza virus is an important pathogen, causing seasonal and sudden pandemics in humans and its avian variants can be devastating for domestic poultry. Moreover, zoonotic transmission of highly pathogenic avian influenza viruses, like H5N1, has been reported regularly. Vaccination is the most promising strategy to control the virus, but traditional vaccines are not very useful in the case of new emerging strains, and therefore, the development of innovative, new-generation vaccines is an urgent need.
We have previously published several papers describing our work on development of DNA vaccine against H5N1 influenza (
The K3/pCI plasmid, containing the full-length cDNA of HA from the highly pathogenic influenza virus strain A/swan/Poland/305-135V08/2006(H5N1, clade 2.2), has been described before as a long variant of DNA vaccine (
Broiler chickens Ross 308 and layer chickens Rosa 1 were purchased from a local commercial brooder on the day of hatching and were maintained at an experimental poultry house under standard bedding conditions. Animals were fed once a day and had free access to water. At the end of the experiment, the animals were humanely euthanized. Chickens were immunized intramuscularly with 60 µg of plasmid in final volume of 100 µl and blood samples were collected from the wing vein. Two doses of vaccine were administered at 7th and 21st days of life.
Specific pathogen-free BALB/c female mice were maintained at the experimental facility at the Mossakowski Medical Research Centre, Polish Academy of Sciences (Warsaw) under a 13-h light/11-h dark cycle with free access to water and standard mouse diet. All groups were immunized intramuscularly with 20 µg of plasmid in final volume of 50 µl and the blood samples were collected from a left ventricle of heart. Two doses of vaccine were administered at 35th and 49th days of life. The schedule of chickens and mice immunization experiments is summarized in Table
Details of the immunization experiments.
Animal model | Experiment Nr (chicken type; dose) | Group | Size ( |
Days of treatments |
||
---|---|---|---|---|---|---|
Immunization | Blood collection | Spleen collection | ||||
Chickens | Experiment 1 (layers; 60 µg) | K3/pCI | 6 | 7, 21 | 21, 28, 35 | – |
3NF/pCI | 6 | |||||
pCI | 2 | |||||
Experiment 2 (broilers; 60 µg) | K3/pCI | 10 | 7, 21 | 21, 35 | – | |
3NF/pCI | 10 | |||||
pCI | 4 | |||||
Experiment 3 (layers; 60 µg) | K3/pCI | 10 | 7, 21 | 35 | – | |
3NF/pCI | 10 | |||||
NFGK/pCI | 10 | |||||
pCI | 3 | |||||
Mice | Experiment 1 (20 µg) | K3/pCI | 6 | 35, 49 | 49, 56, 63 | 63 |
3NF/pCI | 7 | |||||
NFGK/pCI | 7 | |||||
pCI | 2 | |||||
Experiment 2 (20 µg) | K3/pCI | 8 | 35, 49 | 49, 56, 63 | 63 | |
3NF/pCI | 8 | |||||
NFGK/pCI | 8 | |||||
pCI | 7 | |||||
Experiment 3 (20 µg) | K3/pCI | 3 | 35, 49 | 47, 52 | 52 | |
3NF/pCI | 3 | |||||
pCI | 2 |
All efforts were made to minimize suffering. The experiments with chickens were approved by the Second Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, Permit Number 17/2009. The experiments of mice immunization were approved by the Fourth Local Ethical Committee for Animal Experiments at the National Medicines Institutes, Permit Number 03/2014.
The ELISA was performed as described earlier (
Sera from immunized mice were tested for antibodies directed against homologous H5 HA by a one-dilution indirect ELISA using MaxiSorp Surface (Nunc, UK) plates coated with 300 ng of recombinant H5 HA (obtained in the baculoviral system; Oxford Expression Technologies, UK). Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich) was used as the secondary antibody.
Hemagglutination inhibition tests were performed according to the OIE standard procedures using the heterologous hemagglutinating antigen prepared from the low pathogenic H5N2 strain A/chicken/Belgium/150/1999 (DG Deventer, Netherlands). The 25-µl aliquots of serial twofold dilutions (from 1:8 to 1:512) of sera in PBS were added to an equal volume of HA antigen containing four HA units. After incubation (25 min) in V-bottom microtiter plates at room temperature (RT), 25 µl of a 1% suspension of chicken red blood cells was added and incubated for 25 min at RT. HI titers are defined as the reciprocal of the highest dilution of sera that completely inhibited hemagglutination.
Immunized and control mice were euthanized 2 weeks after the boost dose (day 63) and their spleens were harvested. For determining the cytokine production and percentage of Tc and Th, splenocytes were prepared from spleen isolated from three randomly selected mice immunized in Experiment 2. The spleen cells suspensions were washed in RPMI-1640 medium (Sigma-Aldrich) and treated for 5 min with lysis buffer (Becton-Dickinson, Franklin Lakes, NJ, USA) in order to remove red blood cells. To determine the levels of cytokines in culture supernatants (Experiment 1 and Experiment 2), the cells (2 × 106 per well) were incubated in 96-well plates (Corning, NY, USA) in complete RPMI-1640 without any supplement (negative control), with recombinant H5 HA protein (Oxford Expression Technologies, UK) (10 µg/ml) or with concanavalin A (5 µg/ml). Cells were incubated for 72 h (37°C, 5% CO2) and centrifuged (10 min, 1,000 rpm, 4°C). The level of cytokines was quantified in the collected supernatants using the Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit (Becton-Dickinson) according to the manufacturer’s instructions and a FacsVERSE™ flow cytometer (Becton-Dickinson).
For determining the levels of CD8+ (Tc), CD4+ (Th) cells and their corresponding activated subpopulations: CD25+ (Tc), CD69+ (Tc), CD25+ (Th), and CD69+ (Th) splenocytes (1 × 106 per probe) were incubated 30 min on ice with the following monoclonal antibodies (Becton-Dickinson): PerCP rat anti-mouse CD4, FITC rat anti-mouse CD8, APC rat anti-mouse CD25, and FITC rat anti-mouse CD69. After incubation, the cells were washed three times, resuspended in the Stain Buffer (Becton-Dickinson), and examined by flow cytometry using FacsVERSE™ flow cytometer (Becton-Dickinson). The levels of Tc and Th were determined in the Laboratory of Flow Cytometry, Faculty of Biology University of Warsaw.
Microarray expression analysis was performed using the Affymetrix Gene Atlas system according to the manufacturer’s instructions. RNA was isolated from three independent individuals per treatment (K3/pCI and 3NF/pCI) or two independent control individuals (pCI), 3 days after the boosted vaccination; 100 ng of total RNA that passed the initial quality control screen was then prepared for Affymetrix whole transcriptome microarray analysis using the Ambion® WT Expression Kit (4411973). Prepared samples were hybridized to the Affymetrix® Mouse Gene 2.1 ST Array Strip (Affymetrix, Santa Clara, CA, USA). The microarrays were scanned with the Affymetrix GeneAtlas Scanner, and the intensity signals for each of the probe sets were written by Affymetrix software into CEL files. The CEL files were imported into Partek Genomic Suite v 6.6 software with the use of Robust Multiarray Averaging. During this step, a background correction was applied based on the global distribution of the PM (perfect match) probe intensities and the affinity for each of the probes (based on their sequences) was calculated. Then, the probe intensities were quantile normalized (
In order to validate the microarray data, the gene expression level of the selected genes was determined using the same samples of RNA as those used for microarray analysis. The cDNA was obtained from 1.2 µg of total RNA using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) with Oligo(dT)18 primers and was then used as the template in RT-qPCR using Thermo Scientific Luminaris Color HuGreen qPCR master mix (Thermo Sceintific). PCR was performed using the PikoReal™ Real-Time PCR System (Thermo Scientific). Assays contained the cDNA template diluted 20-fold. A 10-min hot-start activation at 95°C was followed by 40 cycles of 15 s of denaturation at 95°C, 30 s of annealing at 60°C, and 30 s of extension at 72°C followed by dissociation analysis (60–95°C). Relative gene expression was calculated according to the 2−ΔΔCt method using TAF8 or PGK1 as the reference gene. The list of used primers is in Table S1 in Supplementary Material.
Non-parametric tests, such as Kruskal–Wallis (for the comparison of multiple groups) or Mann–Whitney
The effectiveness of the three variants of DNA vaccine against H5N1 was tested first in chickens, the natural host of influenza virus. The 3NF/pCI (and NFGK/pCI used only in Experiment 3) plasmids contained κB sites, while the K3/pCI plasmid did not contain such sites. The variants of the DNA vaccine were tested in chickens in three independent experiments, using either layers or broilers (Table
Humoral response of chickens to the tested variants of the DNA vaccine.
In all experiments, the HI test was performed only with sera collected 2 weeks after the booster (day 35) using an H5N2 commercial antigen. The results shown in Figure
The endpoint titers of anti-H5 HA antibody were determined using sera from the final blood collection (35th day) in Experiments 1 and 2 (Figure
The experiments with mice were performed independently to verify the effectiveness of the three tested variants (K3/pCI, NF/pCI, and NFGK/pCI) of the DNA vaccine. The schedule and other details of the experiments are provided in Table
Immune response of mice to the tested variants of the DNA vaccine. The results of the one-dilution ELISA test shown for individuals with medians and the 10th and 90th percentiles indicated for each group, where applicable (Experiments 1 and 2). All sera were diluted 100-fold. Statistically significant differences (
In order to further explore the differences between the mice groups, the levels of cytokines produced by the stimulated
Cytokine levels produced by stimulated splenocytes isolated from the mice immunized with the tested variants of the DNA vaccine.
IFNγ (pg) | TNF (pg) | |
---|---|---|
K3/pCI | ||
3NF/pCI | ||
NFGK/pCI |
The percentage of cytotoxic T cells (Tc) and helper T cells (Th) and their subpopulations in mice spleens.
DNA vaccine | Percentage of the indicated cells in the pull of isolated splenocytes |
|||||
---|---|---|---|---|---|---|
Tc |
Th |
|||||
CD8+ | CD8+CD25+ | CD8+CD69+ | CD4+ | CD4+CD25+ | CD4+CD69+ | |
K3/pCI | ||||||
3NF/pCI | ||||||
NFGK/pCI | ||||||
pCI |
As indicated in Table
As indicated in Table
More differences between the two variants (K3/pCI and 3NF/pCI) of the tested DNA vaccine were revealed by transcriptional profiling of the mice spleens isolated on day 52 (3 days after the booster) from mice immunized in Experiment 3. The analysis was limited to those 180 genes that had at least a ±1.4-fold difference (
Transcriptomic changes in the spleens of mice immunized with K3/pCI and 3NF/pCI in comparison to the group that received the empty vector (pCI).
Several genes (Apol11b, Lyst, HMGA1, Ifi44, IL-1A; the last two genes were not significantly changed in microarrays data) were selected for validation of expression by quantitative real-time PCR. The significant differences between the groups were observed only for Apol11b (upregulated in both K3/pCI and 3NF/pCI group) and Lyst (downregulated only in 3NF/pCI). The results of RT-qPCR analysis are shown in Figure
Summarizing the above results, due to the high individual variability, we do not have strong evidence to conclude that addition of ĸB sites improves the immunogenicity of the vaccine in mouse model.
We demonstrate for the first time in two animal models that the binding sites for NF-κB might improve the efficacy of a DNA vaccine against influenza. It is known that κB motifs can augment nuclear entry of modified vectors and increase the expression level of reported genes in various transfected cells (
Also, numerous studies confirmed that codon optimization to the codon bias of the antigen improves the efficacy of DNA vaccine. Several studies with mammalian cells suggest that increasing the GC content provides better mRNA stability, processing, and nucleocytoplasmic transport. Since we have previously indicated the moderate superiority of GK/pCI vaccine over the K3/pCI vaccine (
The immunological humoral responses induced by the tested plasmids seem to work differently in the used animal models and they slightly vary depending on the experiment. In chickens, the 3NF/pCI worked better than K3/pCI, while a single immunization trial with NFGK/pCI (only in Experiment 3; Figure
In mice, the differences between the variants of DNA vaccine at humoral level were much less pronounced than in chickens, probably because mice are not the natural host of influenza virus. However, in Experiment 1 (Figure
The very low (at the detection limit, not shown) levels of IL-2, IL-6, and IL-10 and absence of IL-4 and IL-17a might be explained by the conditions of the assay and splenocytes cultivation (and induction), which were optimal for IFN-γ, and also by the short half-life of IL-4 (
Comparison of the transcriptional patterns of splenocytes isolated from mice immunized with the two types of vaccine (K3/pCI and 3NF/pCI) with the transcriptional pattern of the control mice (pCI) revealed some differences between the variants. For example, differences in the transcripts level of two microRNA were detected (Table S2 in Supplementary Material): mir181-b was downregulated in the 3NF/pCI, while mir1186 was downregulated in K3/pCI. Interestingly, mir181-b inhibits the expression of importin-α3 that is crucial for translocation of NF-κB from cytoplasm to nucleus. The level of mir181-b is reduced after proinflamatory stimulation, e.g., by TNF-α and the transcription of NF-κB-dependent genes can be activated (
Expression of two genes (Apol11b and Lyst) has been positively verified by RT-qPCR. Information about the function of Apol11b and Lyst, two genes with the expression verified by RT-qPCR, is rather limited. The Apol11b gene, upregulated in both tested groups (3NF/pCI and K3/pCI), encodes the Apolipoprotein L variant specific for the spleen (
Despite extensive work on DNA vaccines, reports describing usage of the binding sites for nuclear factors as stimulators of antigen expression and enhancers of the immune response are quite limited. Results of our study highlight possible positive effects of such modifications on the effectiveness of DNA vaccine. However, extended analysis of the mechanisms responsible for the observed effects is needed before such modifications can be put into practice and used for development of highly immunogenic and safe DNA vaccines.
The experiments were approved by the Second Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, Permit Number 17/2009 (chickens) or the Fourth Local Ethical Committee for Animal Experiments at the National Medicines Institutes, Permit Number 03/2014 (mice). All efforts were made to minimize suffering.
PR performed mice immunization and responses analysis; ASt performed chickens immunization and responses analysis; RS performed transcriptional analysis; PK involved in mice immunization; KB involved in FACS analysis; AG-S and AS conceived and designed the experiments, and prepared the final version of the manuscript. All authors were involved in writing and reviewing the manuscript.
Results described in this work are subject of patent application (decision pending). No other conflict of interest declared.
The authors dedicate this work to the memory of Professor Włodzimierz Zagórski-Ostoja, who was actively involved in its initial stages. They also wish to express their thanks to Prof. Patrick Midoux and Prof. Chantal Pichon (Orléans, France) for their help in designing the vector and the Laboratory of Flow Cytometry (Faculty of Biology University of Warsaw) for flow cytometry analysis of the cells.
The Supplementary Material for this article can be found online at