Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens
- Anna Stachyra†1,
- Patrycja Redkiewicz†1,
- Piotr Kosson2,
- Anna Protasiuk1,
- Anna Góra-Sochacka1,
- Grzegorz Kudla3 and
- Agnieszka Sirko1Email authorView ORCID ID profile
© The Author(s). 2016
Received: 9 June 2016
Accepted: 12 August 2016
Published: 26 August 2016
Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host.
The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides.
The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine.
Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans.
KeywordsChickens DNA vaccine GC content H5N1 Influenza Mice
Vaccines against influenza are traditionally produced from viruses propagated in chicken embryos, however the first formulations with antigens produced in cell lines are also available, for example Flucelvax®. The traditional techniques have a number of disadvantages: (i) the process is slow and inflexible, which hinders a fast reaction in the case of new outbreaks, (ii) capacity is too small to produce enough doses, (iii) workers are exposed to the dangerous live pathogen and (iv) the virus can mutate during propagation. Moreover, highly pathogenic strains are difficult to propagate in eggs in sufficient amounts, due to their harmful effect on the host, and have to be reassorted or genetically engineered. Problematic are also traces of chicken proteins present in formulation, which are common allergens [1, 2]. The DNA vaccines seem to be a very promising alternative with multiple advantages. They are relatively easy and economical in production due to the lack of long-lasting and complicated procedures of antigen multiplication and purification. They can be quickly re-designed and re-constructed in case of sudden new disease outbreaks. They guarantee antigens with native structure, identical with those within infection, containing all posttranslational modifications, since they are produced in vivo in host cells. Moreover, they are safe and no infective form of the pathogen is needed at any step. DNA itself is also more stable in storage and transport than proteins. DNA vaccines induce both humoral and cellular immunological responses, stimulating T cells, antigen presenting cells and antibodies production, ensure broad, long lasting and protective response [3, 4]. Thus, it is not surprising that several clinical trials of DNA vaccines against influenza are now ongoing (http://clinicaltrials.gov/) [5, 6].
The expression level of cDNA encoding an antigen in the cells of immunized host is an important factor influencing the immunological potential of DNA-based vaccines. Manipulations within the coding sequence, such as replacing the rare codons with the synonymous codons preferred by the host organism and avoidance of RNA secondary structures motifs or others unprofitable features have been applied to improve the effectiveness of DNA vaccines against influenza . For example, codon optimization of DNA vaccine based on HAs from A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) not only enhanced its immunogenicity but also might lead to the reduction of the number of required doses . Similar results were also reported for DNA vaccine based on HA derived from the swine influenza virus A/Texas/05/2009 (H1N1) . These authors demonstrated that optimization of the codon bias of HA from H1N1 resulted in stimulation of CD8+ (determined by the high levels of TNF, IFNγ and IL-2) and in elevated level of antibody production. Recently, also immunization of ponies (mixed breeds of Shetland blood, Welsh blood, Florida swamp pony blood) with monovalent or trivalent DNA vaccines (with mammalian preferred codons) encoding HAs from different strains of H3N8 equine influenza was reported . The vaccine was administered to ponies that were subsequently challenged with the homologues virus. The degree of protection, virus shedding and clinical symptoms after infection were significantly reduced in all immunized groups compared to the negative control. Moreover, a moderate level of cross response was obtained in the group that received the trivalent formulation.
Avian influenza is a serious and highly infectious disease of poultry and other bird species, caused by influenza viruses which can be also transmitted to humans causing high mortality [11, 12]. Therefore, development of effective vaccines against avian influenza is very important. In birds, higher effectiveness of the DNA vaccine based on the HA variant with codons optimized for chicken usage, where the optimized gene shared about 75 % nucleotides with the wild type gene, has been reported by several independent research groups. The examples include chicken  and Japanese quails  immunization by different variants of H5 HA. The authors mentioned several possible reasons of the observed superiority of the modified plasmid, such as increased expression due to usage of the chicken optimized codons, increased mRNA stability due to increased GC content and increased level of CpG motifs that could act as an adjuvant of immunological responses . In contrast, no significant seroconversion differences between the groups immunized with the optimized and non-optimized variants were observed in the case of the DNA vaccine based on HA from the low pathogenic H6N2 virus . The authors observed high inter-individual variation, possibly due to poor efficiency of the delivery method and/or the huge biological variation of individual responses. The H5 HA variants optimized for human preferred codons were also tested. For example, a large set of different HA optimized for human preferred codons was tested in mice and chickens and proven to elicit robust protective immune responses against a broad range of H5 influenza strains . Animals received several multivalent combinations of DNA vaccines. Responses were tested by HI and virus microneutralization tests with homologous and heterologous antigens, as well as by the challenge experiment. The obtained results indicated protection against heterologous strains of highly pathogenic avian influenza H5N1 after vaccination with two doses of DNA vaccine. Another interesting approach was applied by the researchers who used the consensus sequence of H5 HA based on the sequences from 467 different H5 strains, optimized it for mammalian expression (using human preferred codons) and observed not only its high expression level but also strong protective immune response in vaccinated laboratory animals .
Most above-mentioned studies confirmed that codon optimization to the codon bias of the host improves the efficacy of DNA vaccine. However, several studies in mammalian cells suggest that increasing the GC content provides better mRNA stability, processing and nucleocytoplasmic transport [18–21]. Because the distribution of GC content among genes is similar in mammals and birds (Additional file 1: Figure S1), we hypothesized that GC content optimization might also lead to improved expression and immunogenicity in birds. The DNA vaccine prepared according to such criterion could be effective in many types of hosts, its design would be simplified and the obtained effects more universal. Therefore, the goal of this study was to evaluate the immunogenicity of the variant of DNA vaccine containing nearly 100 % of codons with GC at the third position in two model animals, mice and chickens, and comparing it to the other vaccine variants. All tested vaccine variants were based on HA from the highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1).
Plasmids used for DNA vaccination
The HAw/pCI, K3/pCI and GK/pCI plasmids were used for DNA vaccination. The HAw/pCI plasmid contains the nucleotide sequence identical to the region encoding the full-length HA from A/swan/Poland/305-135V08/2006 (H5N1). The K3/pCI and GK/pCI plasmids contain two different nucleotide sequences encoding the same H5 HA protein as HAw/pCI (with the leader peptide) but without the proteolytic cleavage site (341-RRRKKRR-347) between HA1 and HA2 subunits. The sequence encoding HA, present in K3/pCI, was optimized for domestic chicken (Gallus gallus) and the codon adaptation index (CAI) reached 0.91. In contrast, the sequence encoding HA, present in GK/pCI was not optimized to any codon bias but it was was modified by changing the nucleotides present in the third positions of the codons to either guanine (G) or cytosine (C). The cDNA of K3 and GK were synthesized by GeneScript (USA; http://www.genscript.com/). Comparison of the HA sequences is shown in Additional file 2: Figure S2. The inserts were cloned into MluI and SalI restriction sites of the pCI expression vector (Promega, Wisconsin, USA) downstream of the cytomegalovirus (CMV) promoter and upstream of the SV40 late polyadenylation signal. Plasmids were propagated in DH5α strain of Escherichia coli and isolated using NucleoBond® PC 10000 EF Giga-scale purification kit (Macherey-Nagel, Düren, Germany).
Transfection of mammalian cells
The mouse myoblast cells (2x105 cells; C2C12 line) were transfected with 2 μg of HAw/pCI, K3/pCI, GK/pCI or pCI using Lipofectamine® 3000 Reagent (ThermoFisher Scientific, Waltham, USA) as described by the manufacturer. After 48h the cells were scraped off into the RIPA buffer (ThermoFisher Scientific, Waltham, USA), transferred to the 1.5 ml tubes and frozen in liquid nitrogen, following thawing at 37 °C (three times). The homogenates were centrifuged at 10 000× g for 8 min at 4 °C and the equal amounts of protein extract were analyzed by SDS-PAGE (Nu-Page™ 4-12 % Bis-Tris gel, Invitrogen™, Basel, Switzerland) and Western blotting. The nitrocellulose membranes were blocked in 5 % milk in 1× TBS buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 % Tween 80) and incubated for 1.5h with primary antibody anti – HA (H5) (1:500, ImmuneTechnology, USA), or anti- GAPDH (1:20000, Sigma, St. Louis, USA) and for 1h with secondary antibody (anti-rabbit or anti-mouse IgG (whole molecule) − alkaline phosphatase antibody; Sigma, St. Louise, USA). The enzymatic color reaction was generated using NBT/BCIP Stock Solution (Roche, Switzerland). The bands intensity was compared using the Image J (https://imagej.nih.gov/ij/).
Immunization of animals
Number of animals used in immunization experiments
Number of animals per group
Experiment 1 (Broilers)
Experiment 2 (Layers)
Experiment 3 (Broilers)
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. 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 experiments with chickens were approved by the Second Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, Permit Number 17/2009.
Mice: 96-well flat-bottom plates (MaxiSorp Surface, Nunc, UK) were coated with 300 ng of purified recombinant H5 HA (A/swan/Poland/305-135V08/2006, H5N1) (derived from a baculovirus system (Oxford Expression Technologies, UK) at 2–8 °C overnight. After removing the coating buffer, the plates were washed three times with 1× PBST (phosphate buffered saline with 0,05 % of Tween-20) and blocked with 2 % of BSA-PBST at 37 °C for 90 min. After 2 washes, the 100-fold in Experiment 1, Experiment 3 and Experiment 4 and the 50-fold (49d) or 200-fold (56d and 63d) in Experiment 2 diluted sera samples were added and incubated at 2–8 °C overnight. Next day, after 4 washes, plates was incubated for 1h at 37 °C with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma Aldrich, St. Louise, USA). The enzymatic color reaction was performed using alkaline phosphatase yellow (pNPP) liquid substrate (Sigma, St.LouiseUSA), stopped with 3M NaOH and measured (OD405) using a Synergy/HT microplate reader (BioTek Instruments, Inc.).
Chickens: 96-well flat-bottom plates (MediSorp Surface, Nunc, UK) were coated with the same antigen as above. After removing the coating buffer, the plates were washed four times with 1× PBST and blocked with 2 % of BSA-PBST at 37 °C for 90 min. Following 2 washes, the 200-fold diluted sera samples were added and incubated at 2–8 °C overnight. Next day, after 5 washes plates were incubated for 1h at 37 °C with peroxidase-conjugated goat anti-chicken IgY (Life Technologies, USA). The enzymatic color reaction was generated using TMB substrate (Sigma, St.Louise, USA), stopped with 0.5M H2SO4 and measured (OD450) as above.
Antibody endpoint titers
For determination of IgY endpoint titers two-fold serial dilutions of chicken sera collected on day 35 (in range from 10−3 to 10−6) were made and analyzed using the ELISA protocol. Based on OD450 values the absorption curves were made and the endpoint titers were determined using Gen5 Data Analysis Software (BioTek Instruments, Inc.).
Hemagglutination inhibition (HI)
HI tests were performed according to the OIE standard procedures using the commercially available hemagglutinating antigen prepared from low pathogenic H5N2 strain A/chicken/Belgium/150/1999 (DG Deventer, Netherlands) with 96 % protein sequence similarity to the vaccine antigen. For the HI test, serum to be tested was serially two-fold diluted (1:8 to 1:512) in 25 μl of PBS in V-bottom microtiter plates and an equal volume of HA antigen containing 4 HA units was added. After incubation at room temperature (RT) for 25 min, 25 μl of a 1 % suspension of hens’ red blood cells was added and incubated for 25 min at RT. HI titers are shown as the reciprocal of the highest dilution of sera that completely inhibited hemagglutination.
Cytokine production assay
Immunized and control mice were euthanized two weeks after boost dose (day 63) and their spleens were harvested. The spleen cell suspensions from the pooled two spleens (from two randomly selected mice from the same group) were washed in RPMI-1640 medium (Sigma, USA) and treated for 5 min with the Lysis buffer (BD, Franklin Lakes, USA) in order to clear red blood cells. To determine the amount of cytokines in culture supernatants, splenocytes (2x106 per well) were incubated in 96-well plates (Corning, Corning, USA) with complete RPMI-1640 with 10 μg/ml of recombinant H5 HA protein purified from the baculovirus system (Oxford Expression Technologies, England), 5 μg/ml Concavaline A (Con A) or medium alone (see above). Cells were incubated for 72h (37 °C, 5 % CO2) and centrifuged (10 min, 1000 rpm, 4 °C). The level of cytokines was quantified in the collected supernatants using Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit (BD, Franklin Lakes, USA) according to the manufacturer’s instructions and the FASCCalibur™ flow cytometer (BD, Franklin Lakes, USA).
Non-parametric tests, such as Kruskal-Wallis (for comparison of multiple groups) or Wald-Wolfowitz, Kolmogorov-Smirnov and Mann-Whitney U (for comparison of two groups) that are components of Statistica 12 (StatSoft, Poland) were used to evaluate the statistical differences. The groups were considered significantly different if at last one of the test was positive (p < 0.05).
Verification of the HA expression cassettes in mammalian cells
Comparison of the effectiveness of HAw/pCI and K3/pCI in two animal models
Mice response to the optimized variants of DNA vaccine
Chickens response to the optimized variants of DNA vaccine
Next, the endpoint titers of anti-H5 HA in the sera collected two weeks after the booster (on day 35) were assayed and, in order to facilitate the interpretation of data, they were arbitrarily divided into four categories: high (>105), medium (104-105), low (103-104) and very low (<103) (Fig. 6c). In layers, none of the probes from the K3/pCI group reached the end-point titer above 105 and 33 % of probes did not exceed the titer above 103, in contrast to the 33 and 17 % of such probes, respectively, in the case of GK/pCI group. The highest titer of layers’ sera from K3/pCI and GK/pCI group was 7 × 104 and 4 × 105, respectively. The endpoint titers of the broilers’ sera indicated less variability within the groups and the highest titers were more similar (both about 2 × 105). In broilers, 40 % of the probes from the K3/pCI group had titers above 105 and all of them were above 103, while all sera from the GK/pCI group had titers above 104, including 33 % with the titer above 105.
The results of hemagglutination inhibitions (HI) test seem to confirm a slightly better performance of the GK/pCI vaccine over the K3/pCI vaccine in both chicken experiments, however the differences are not statistically significant (Fig. 6d).
DNA vaccines containing the optimized variants of H5 HA gene induced strong and specific immune responses in mice and chickens. In both animal models the slight superiority of GK/pCI over K3/pCI was observed. The observed differences were frequently statistically significant. Changes within GK did not regard the codon usage preference in any particular organism but the key was maximization of the GC content at the third coding position of HA (43, 65 and 99.8 % in HAw/pCI, K3/pCI and GK/pCI, respectively). This study was inspired by the previous reports indicating that an increased GC content provides better mRNA stability, processing and nucleocytoplasmic transport [18, 20]. In fact, our results can be explained and are in full agreement with the above literature data. We started with verification of the modified cassettes by monitoring of the level of HA protein produced in mouse muscle cells (C2C12) transfected with K3/pCI and GK/pCI. Indeed, about 15–30 % higher level of HA protein production was observed in cells transfected with GK/pCI than with K3/pCI, which corresponds well with apparently higher immune responses to GK/pCI than to K3/pCI in the immunized animals. The correlation between in vitro expression in transfected cells and immunogenicity of DNA vaccine was also observed by others [24, 25].
Little is known about the timing of cytokine production after immunization. We investigated the profiles of cytokines in the supernatants from the cultured, stimulated with H5 HA for 72h, mice splenocytes that were isolated from the spleens of the immunized animals. In the supernatants we confirmed the presence of five of seven tested cytokines. The levels of IFN-γ, TNF and IL-6 were higher in the group immunized with GK/pCI than with K3/pCI. This result suggests that codon optimization affects both branches of immune responses, humoral (IL-6) and cellular (IFN-γ, TNF) what was previously reported in studies with HIV and HPV DNA vaccine [26, 27]. The lower levels of IL-2 and IL-10 in GK/pCI than K3/pCI group is unclear and need further investigation. The lower level of IL-2 was also observed by Tenbusch et al. in stimulated CD4+ from mice immunized with DNA vaccine containing the optimized sequence for the HA from H1N1 . We did not detect IL-4 nor IL-17A. The lack of IL-17A might be explained by the high concentration of IFN-γ negatively regulating the induction of Th17 cells . The lack of IL-4 might be explained by the conditions of the assay and splenocytes cultivation (and induction) which were optimal for IFN-γ but not IL-4 detection due to the short half-life of the letter . Additional analysis of mice sera (data not shown) indicated that although the level of IgG2a (one of two major isotypes of antibodies) was rather similar in both groups, the level of IgG1 was slightly higher in GK/pCI than in K3/pCI. This result might suggest that the elevation of immune response in GK/pCI group concerned mostly the elevation of Th2 type, however these aspects of the response to the vaccine variants require more studies.
Better efficacy of GK/pCI than K3/pCI observed in chickens is in contrast with the results by Rao et al.  who reported that in chicken genome the GC content at the third coding position is negatively correlated with the expression level and that it is not correlated with the maximum expression level. Based on their own analysis, the authors stated that the GC content in genes (general, not only in the third codon positions) could explain only approximately 10 % of the variation in gene expression. According to Kudla at al.  the efficient transcription or mRNA processing is responsible for the high expression of GC-rich gene, while other researchers (for example [31, 32]) assumed that the increased expression of codon–optimized genes was caused by the more efficient translational mechanism. The high effectiveness of the variant with nearly 100 % codons with GC at the third position in both model animals (regardless of the codon usage preferences) suggests that the first hypothesis might be correct.
Interestingly, the immunization was generally more effective in broilers than in layers. The results of ELISA test, as well as of HI test were less variable within the broiler groups (70–88 % positives). Immunization of the layers type chickens gave slightly lower and more variable antibody levels and slightly worse results in the HI test (Fig. 5c). On average, the endpoint titers were lower in layers than in broilers, particularly in K3/pCI groups. Many factors can disturb the effectiveness of immunization and the chickens might respond in a very individual way, as observed by others, too . DNA uptake by the target cells, preceded by penetration of sufficient area of tissue after injection seems to be crucial. It is worth to emphasize that the dose chosen for chickens’ immunization was suboptimal for better visualization of the expected differences in immune response. Differences in the strength and dynamic of broilers’ and layers’ responses can be linked to the different genetic background of two used chicken types, differences in their metabolism, growth and development which are results of intensive genetic selection . Chickens are not so popular animal model as mice in immunological studies, still considerable number of publications about chicken immunization with DNA vaccine, especially with avian influenza antigens are available . Most of such experiments were performed with specific pathogen free White Leghorn chickens. In this study we used birds, which are popularly used in Poland for the commercial purposes: the broiler line Ross 308 and the layer line Rosa 1 (a hybrid of Rhode Island and Sussex) and kept them in standard commercial conditions, which allow us to observe natural reactions to the immunizations. Moreover, to our knowledge this is the first report on comparison of the layers’ and the broilers’ humoral responses to the DNA vaccine. Some papers comparing immunological responses of broiler and layer types are available [34–36], however in these experiments conventional vaccines against Salmonella sp. were used, or synthetic peptide antigen, not originated from any poultry disease. Similarly to our results, differences in responses between broilers and layers have been previously reported.
In summary, our results strongly suggest that the enhancement of GC content in the third positions of the codons is a promising strategy for development of DNA vaccine that could be highly effective in a broad range of target species, such as birds and mammals, including humans.
We dedicate this work to the memory of Professor Włodzimierz Zagórski-Ostoja, who was actively involved in its initial stages.
This study was funded by the National Center for Research and Development (EC Innovative Economy Program POIG.01.01.02-00-007/08) and in part by Grant No. PBS2/A7/14/2014 from the National Centre for Research and Development. GK was supported by the Wellcome Trust (097383) and by the Medical Research Council.
ASt and AP conducted the chicken experiments, PR and PK conducted the mice experiments, ASi, AG-S and GK participated in study design and data analysis. All authors participated in manuscript and figures preparation, have read and approved the final manuscript.
The authors declare that they have no competing interests. Part of presented results is a subject of pending patent application P-411230 (Poland).
Ethics approval and consent to participate
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments of mouse immunization were approved by the Fourth Local Ethical Committee for Animal Experiments at the National Medicines Institutes, Permit Number 03/2014. 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.
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