Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses

Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5- and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9- and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15- vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens

Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene

Fowl pox virus (FPV) has been modified to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to co-express a novel host cytokine from such recombinants and characterize its immune response. In this study, previously constructed H5-recombinant Fowl pox viruses co-expressing host IL-15 (rFPV) was inoculated into specific-pathogen-free (SPF) chickens. T-lymphocyte populations and selected cytokine expression namely IL-15 and IL-18 of the vaccinated chickens were evaluated to add the relatively limited knowledge of chicken IL-15 cytokine gene compared to that of mammalian. It is hypothesized that vaccination with H5-recombinant Fowl pox viruses co-expressing IL-15 gene is able to show a higher population percentage of CD4+ and CD8+ T lymphocytes, and high IL-15 and IL-18 expressions, compared to H5-recombinant vaccines alone, in chickens. Prior to in vivo characterization, recombinant viruses were propagated in Chicken Embryonic Fibroblast (CEF) primary cell line. Stability of H5 gene from influenza strain A/Chicken/Malaysia/5858/2004 (1695 kb), and IL-15 (695 kb) gene integrations was confirmed by using Polymerase Chain Reaction (PCR) with specific primers after three passages. Propagations and plaque assays were done until desired titres of recombinant viruses were obtained. Parental (FP9 wild-type) and recombinant virus vaccines (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. The immunogenicity of the recombinant viruses was analyzed based on evaluation of T-lymphocytes cell population via flow cytometry, from Peripheral Blood Mononuclear Cell (PBMC) of 14 and 28-days-old vaccinated chickens. Chickens inoculated with rFPV/H5/IL-15 had a higher increased in CD4+ T cells population relative to rFPV/H5 in both time points. However, the result showed that rFPV/H5/IL-15 was not significant (P>0.05) in inducing CD8+ T cells. In general, the percentage of CD4+ and CD8+ lymphocytes cell population in chickens immunized with rFPV/H5/IL-15 were statistically higher compared to chickens immunized with rFPV/H5 and FP9 wild-type virus (P<0.05). Specific gene expressions of SPF chickens inoculated with rFPV were analyzed by quantitative real-time PCR (qRT-PCR), following extraction of spleen from 14-day-old SPF chickens at days 2, 4 and 6 post-infection. Two target genes chosen were IL-15 and IL-18 genes. The rFPV/H5/IL-15 group showed an increased level of IL-15 and IL-18 genes expression up to 2 and 3.5 folds, respectively, within 6 days post-vaccination, compared with other inoculated groups. rFPV/H5 group showed an increased level of IL-15 gene expression at day 2 and maintained at day 4 until day 6, while the IL-18 expression was decreasing within 6 days. Overall, the FP9 wild type group showed a low cytokine expression level as compared to the recombinant virus groups. While histopathology results showed successful vaccination of rFPV into chicken cells, weekly weighing suggested that inoculation with rFPV might not influence any weight changes. In summary, this study showed modulation immunogenicity of FP9 Wild Type, rFPV/H5, and rFPV/H5/IL-15, with rFPV/H5/IL-15 being the best vaccine candidate compared to others

Negligible effect of chicken cytokine IL-12 integration into recombinant fowlpox viruses expressing avian influenza virus neuraminidase N1 on host cellular immune responses

In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 105 p.f.u. ml−1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate

Verification of H5 and IL-15 gene integrations in recombinant fowlpox viruses

Introduction of the chicken cytokine gene might trigger a stronger cellular mediated immune response in recombinant fowlpox viruses already carrying the avian influenza H5 gene. In order to ascertain that integrated H5 and IL-15 genes are stable in the previously constructed recombinant fowlpox viruses, PCR analyses were done after a few viral passages. Wild type fowlpox virus, recombinant fowlpox viruses co-expressing avian influenza H5 (rFWPV/H5) and recombinant fowlpox viruses co-expressing avian influenza H5 and chicken IL-15 cytokine (rFWPV/H5) were propagated in CEF cell culture. DNA genomic extractions were done prior to PCR analysis. Primer set targeting H5 and IL-15 confirmed the deduced sizes of both genes. Flanking primers further verified the stable integration of H5 and IL-15 genes in both recombinant viruses. This study confirms that H5 and IL-15 genes were stably present rFWPV/H5 and rFWPV/H5/IL-15. The current results allow future in vitro and in vivo characterisation of the recombinant viruses in triggering humoral and cellular immune responses

Enhancement of bioelectricity production from soil microbial fuel cell (SMFC) by additional glucose, nutrient broth and escherichia coli bacteria

Soil Microbial Fuel Cell (SMFC) is a device that using bacteria in soils as a biocatalyst. These bacteria, called exoelectrogenic bacteria are oxidizing organic substrates to release electrons, which then harvested in an external circuit to produce bioelectricity. Despite all the potential, the bioelectricity production from soils is still low and its relation with SMFCconditions is uncertain. Hence, the main objective in this study is to enhance and stabilize the bioelectricity production of SMFC by additional glucose, nutrient broth and Escherichia coli (E. coli) as exoelectrogenic bacteria. A number of factors of SMFC performance were first identified to be preliminary investigated, that is the type of electrode, water addition to soil and distance between anode to cathode. It has been established in this study to use SMFC with the configuration of 9.5 cm in diameter and 15 cm height of the plastic container, with the 12 cm distance between carbon felt of anode and cathode. The electricity produced was measured by using a multimeter in term of voltage reading (mV). From this study, the highest bioelectricity produced was obtained from SMFC using nutrient broth with a maximum voltage of 700 mV. It has found that the additional E. coli bacteria did not increase the bioelectricity production. The use of E. coli needed to be combined with nutrient broth in order to achieve high and stable bioelectricity. It can be suggested that the indigenous bacteria that exist in the soils possibly played the role in producing bioelectricity