The in vivo studies using SPF chickens were conducted after confirming that all the plasmid constructs were able to express the desired proteins and were transcriptionally active in the in vitro expression analysis. In addition, the transgenes were stable over a period of 6 weeks after the booster dose, thus implying the potential that the plasmid DNAs encoding the target proteins could be expressed over a sustainable period.
Chickens inoculated with pDis/H5 + pDis/IL-15 at 4 weeks post inoculation in Trial 1 (Table 4) and Trial 2 (Table 5) showed a 2.82 fold and 4.87 fold increase in the HI antibody titer respectively, compared to pDis/H5 expressing the H5 alone (P < 0.05), suggesting that IL-15 plays a role in enhancing the humoral immune response. This finding was comparable with several studies conducted in different animal models where the recombinant vaccinia virus vaccine with integrated IL-15 expressing gp160 of human immunodeficiency virus generated a more robust and durable antibody response compared to gp160 alone . In another study, the recombinant vaccinia virus-based pentavalent H5N1 AI vaccine adjuvanted with the human IL-15 induced stronger neutralizing antibodies against the AIV H5 in mice . Another study demonstrated that the recombinant protein from the Eimeria acervulina in combination with the chicken IL-15 against coccidiosis which exhibited significantly greater serum IgG antibody levels than with the recombinant protein alone in ovo.
This study also showed an increased in the HI titer by the pDis/H5 + pDis/IL-18 at 4 weeks post inoculation in Trial 1 (Table 4) and Trial 2 (Table 5) by 2.14 fold and 2.00 fold respectively, compared to the pDis/H5 expressing the H5 alone (P < 0.05). Several reports have shown that the plasmid encoding IL-18, which is used as an adjuvant in mammalian vaccines, promote high antibody titers [20, 21]. The recombinant chicken IL-18 has been shown to significantly enhance antibody responses to the Clostridium perfringens α-toxoid and NDV antigens . Higher HI titers induced in chickens immunized with the Newcastle disease vaccine co-administrated with chicken IL-18 further confirm the immunostimulatory activities of chicken IL-18 in vivo.
The highest HI GMT of 16 ± 0.00 was observed in SPF chickens at 4 weeks post inoculation after two pDis/H5 + pDis/IL-15 inoculations were given in Trial 2. This is comparable to the studies conducted by our group where chickens immunized intramuscularly with the H5 DNA vaccine with genetic adjuvants, such as MDP-1, Esat-6 and HSP70 of the Mycobacterium tuberculosis, developed a higher HI titer compared to the H5 DNA vaccine alone [24–26]. However, all the previous studies required three vaccinations to achieve a comparable HI titer. Generally, the HI titer obtained in the present study was low compared to the following studies using inactivated AI vaccine. Chickens vaccinated with inactivated AI vaccine with HI GMT of 10 to 40 were associated with the prevention of mortality and HI GMT of 80 or more were associated with protection for mortality . However, a previous study has shown that although the HA gene DNA vaccine induced a low HI antibody level after two DNA injections, immunized chickens were still protected against the lethal HPAI virus challenge , suggesting the role of other immune components, particularly in cellular immunity. Previous studies have shown that although the antibody against HA played a role in neutralizing viral infectivity, cellular immunity plays an important role in viral clearance [29, 30].
The flow cytometry results for both trials (Table 6 and Table 7) demonstrated that pDis/H5, pDis/H5 + pDis/IL-15 and pDis/H5 + pDis/IL-18 were able to trigger higher CD4+ T cell production upon inoculation. The co-administration of SPF chickens with the pDis/IL-15 adjuvant was able to induce a higher production of CD4+ T cells than the pDis/H5 group (P < 0.05). These findings were consistent with previous reports that IL-15 has profound effects on the proliferation and survival of CD4+ T cells . On the other hand, the present study revealed that pDis/IL-15 was not significant (P > 0.05) in inducing CD8+ T cells (Table 6). Nevertheless, in mammalian studies, IL-15 has been shown to support the growth of CD8+ T cells [32, 33]. As shown in Figure 6 and Table 8, the presence of the IL-15 transcript in the spleen and muscles of chickens indicated that chicken IL-15 was able to express in vivo and the low level of CD8+ T cells was not due to the absence or low expression of the IL-15 protein.
The flow cytometry results in Trial 2 (Table 7) demonstrated that the co-administration of pDis/H5 + pDis/IL-18 was able to trigger a higher CD4+ T cell proliferation than the pDis/H5 group (P < 0.05). These findings were consistent with previous reports that IL-18 yielded stronger CD4+ T cell responses in immunized mice  and chickens  thus enabling DNA vaccines to achieve enhanced immune responses. On the other hand, the reports also showed that the co-administration of the IL-18 plasmid as an encoded adjuvant yielded stronger CD8+ T cells . However, both trials in the present study revealed that pDis/IL-18 was not significant (P > 0.05) in inducing CD8+ T cells in the inoculation group. This indicated that IL-18 did not influence CD8+ T cell proliferation significantly, at least not in the PBMC samples at 2 weeks and 5 weeks post inoculation in Trial 1 and 3 weeks and 6 weeks post inoculation in Trial 2.
This study also suggests that the age of the SPF chickens at the time of inoculation could have influenced the immune response of the chickens, where chickens inoculated when they were 14 days old showed a higher HI antibody titer compared to those vaccinated when they were a day old (Table 4 and 5). This finding is comparable to studies which have shown that the priming of the SPF chickens at different ages played a significant role in determining the immune responses [36, 37]. The results in this study showed that the transgenes were stable over a period of 6 weeks after the booster dose, thereby implying the potential that the plasmid DNAs encoding the target proteins could be expressed over a sustainable period. A previous study has shown that chickens vaccinated with the DNA vaccine resulted in the distribution of the target genes in the heart, lungs, liver, spleen and kidneys .
The cytokine expression levels of the immunized chickens were evaluated using GeXP analysis. The GeXP assay was designed originally for a high throughput and differential assessment of a multiplexed expression profile by a single RT-PCR . The assay has a high dynamic linear range and improved sensitivity and specificity compared to other platforms . The co-administration of the H5 DNA vaccinated SPF chickens with the pDis/IL-15 adjuvant was able to induce higher IL-15 gene levels than the control group (P < 0.05). However, only low levels of the IL-18 gene were observed among the treatment groups compared to the control group. Other cytokine genes including the TGF-β, GMCSF, IL-4, TNFSF13B, IL-1β, IFN-γ, IL-12β showed low levels of gene expression following the H5 DNA vaccination, probably due to the fact that the 56-day-old chicken spleens were collected at the end of the experiment.
In this study, we evaluated the immuno-regulatory effects of the co-administration of pDis/H5 + pDis/IL-15 or pDis/IL-18 via assessed on the peripheral blood CD4 and CD8 population and the cytokine expression levels. These results provide evidence that the pDis/IL-15 adjuvant was able to increase the CD4+ T cell population and stimulate the expression of IL-15 which might play a part in immunity against influenza via the induction and maintenance of CD8+ T cell memory, and providing help to B cells for antibody production. However, studies on the biological activities of IL-15 and IL-18 using the in vitro recall assay or evaluation on the regulation of cytokines on the NK cell (CD3-/CD8+) activity and interferon-γ production should be further appraised in the future together with the challenge of the H5N1 virus against the vaccinated chickens. These studies can contribute to the understanding on the role of the pDis/IL-15 adjuvant as a protection against the AIV.