Development of DNA and Oral Vaccines Against Chicken Anemia Virus

Chicken anemia virus (CAV) belongs to the genus Gyrovirus of Circoviridae family, which causes an economically important disease in the poultry industry worldwide. Commercially available vaccines against CAV infection which are based on nonattenuated virulent CAV propagated in chicken embryos or attenuated live vaccine cannot be used in chickens before 8 weeks of age and within 21 days of slaughter and sometimes spreading of the modified viruses to young chickens may cause disease. Therefore, the development of DNA and oral vaccines against this virus was considered in the current study. For development of DNA vaccine, the VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBud-VP1, and pBudVP2-VP1. In vitro and in vivo studie showed that co-expression of VP1 with VP2 is required to produce the essential neutralizing form of VP1 to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old specific pathogen free (SPF) chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. Serum antibody titers against CAV were determined 10 days after vaccination by ELISA. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive antibody titer (1853) against CAV. Furthermore, proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 (78.2 pg/ml) and IFN-γ (534.5 pg/ml) were detected in the pBudVP2-VP1-vaccinated group indicating the induction of cell-mediated immune response in the vaccinated chickens. These results suggest that the recombinant plasmid pBudVP2-VP1 can be used as a potential DNA vaccine against CAV infection. To enhance CAV-specific immune responses, the use of the VP22 gene of Marek’s disease virus type-1 (MDV-1) linked to the CAV VP1 gene was also investigated. This was achieved by constructing a recombinant DNA plasmid, namely pBudVP2-VP1/VP22 encoding the fusion protein VP1/VP22 synchronously with the CAV VP2 and testing its effectiveness in specific pathogen free chickens. Chickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV,VP1-stimulated proliferative induction of splenocytes and also higher levels of IL-2 and IFN-γ when compared to the chicken vaccinated with pBudVP2-VP1 (P<0.05). These observations showed that the MDV-1 VP22 is capable of enhancing the potency of DNA vaccine against CAV when fused with CAV VP1 gene expressing simultaneously with CAV VP2. In the second part of this study, Lactobacillus acidophilus (ATCC 53672) carrying the VP1 protein of CAV was used as live delivery vehicles for oral immunization against CAV. The binding domain of AcmA anchor protein of Lactococcus lactis MG1363 were used to display the VP1 protein of CAV on the surface of Lb.acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expressionvector, pBudCE4.1 to construct plasmids pETacma1 and pETacma2, respectively. Thereafter, the VP1 gene of CAV was fused to the acmA sequences and the CAV VP2 gene was cloned into the second multiple cloning site on the same vector before transformation into Escherichia coli BL 21 (DE3). The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of Lactobacillus acidophilus. Whole cell ELISA and immunofluorescence assay showed binding of the fusion proteins containing the CAV VP1 protein on the surface of the cells. The lactobacilli cells carrying the VP1 protein were used to immunize SPF chickens through oral route. The presence of anti-CAV antibodies was detected by ELISA. The immunized chickens showed significantly (P<0.05) higher level of neutralizing antibody against CAV compared to those from the controls. VP1-specific proliferative response was also observed in splenocytes of the chickens after oral immunization. Furthermore, high levels of Th1 cytokines, IL-2 and IFN-γ were d tected in the immunized chickens. These results showed that the lactobacilli cells carrying the VP1 protein of CAV could induce immune response against CAV suggesting lactobacilli as live delivery vehicle for oral immunization purposes

Improving the potency of DNA vaccine against Chicken Anemia Virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) Type-1 VP22 protein

Studies have shown that the VP22 gene of Marek’s Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV). The VP1 and VP2 genes of CAV isolate SMSC-1 were amplified and inserted into eukaryotic co-expression vector, pBudCE4.1 to construct pBudVP2-VP1. We also constructed pBudVP2-VP1/VP22 encoding CAV VP2 and the VP22 of MDV-1 linked to the CAV VP1. In vitro expression of the genes was confirmed by using RT- PCR, Western blot and indirect immunofluorescence. The vaccines were then tested in 2-week-old SPF chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. After in vivo expression studies, immune responses of the immunized chickens were evaluated pre- and post-immunization. Chickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV and also proliferation induction of splenocytes in comparison to the chickens vaccinated with pBudVP2-VP1. Furthermore, the pBudVP2-VP1/VP22-vaccinated group showed higher level of the Th1 cytokines IL-2 and IFN-g. This study showed that MDV-1 VP22 gene is capable of enhancing the potency of DNA vaccine against CAV when fused with the CAV VP1 gene

Improvement of SCP production and BOD removal of whey with mixed yeast culture

This research emphasizes on single cell protein (SCP) production and Biochemical Oxygen Demand (BOD) removal from whey with mixed yeast culture. For this purpose, 11 yeast strains were isolated from dairy products (M1-M11) and the strains were identified by morphological and physiological properties. These yeast strains were tested for their ability to reduce the BOD and to produce SCP from whey. Among these strains, K. lactis (M2) had the most SCP production from whey with the yield of 11.79 g/l. Ammonium sulphate as nitrogen source had an increasing effect on biomass yield. The mixed culture of the isolated yeast strains with Saccharomyces cerevisiae was used in order to increase the biomass yield and BOD removal. The highest biomass yield (22.38 g/l) and reduction of initial BOD from 30000 to 3450 mg/l were obtained with the mixed culture of K. lactis (M2) and S. cerevisiae

Lactobacillus acidophilus as a live vehicle for oral immunization against chicken anemia virus.

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens

Improvement of SCP production and BOD removal of whey with mixed yeast culture

This research emphasizes on single cell protein (SCP) production and Biochemical Oxygen Demand (BOD) removal from whey with mixed yeast culture. For this purpose, 11 yeast strains were isolated from dairy products (M1-M11) and the strains were identified by morphological and physiological properties. These yeast strains were tested for their ability to reduce the BOD and to produce SCP from whey. Among these strains, K. lactis (M2) had the most SCP production from whey with the yield of 11.79 g/l. Ammonium sulphate as nitrogen source had an increasing effect on biomass yield. The mixed culture of the isolated yeast strains with Saccharomyces cerevisiae was used in order to increase the biomass yield and BOD removal. The highest biomass yield (22.38 g/l) and reduction of initial BOD from 30000 to 3450 mg/l were obtained with the mixed culture of K. lactis (M2) and S. cerevisia

Development of tat - conjugated dendrimer for transdermal DNA vaccine delivery

PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page

Cytotoxicity and immunological responses following oral vaccination of nanoencapsulated avian influenza virus H5 DNA vaccine with green synthesis silver nanoparticles.

DNA formulations provide the basis for safe and cost effective vaccine. Low efficiency is often observed in the delivery of DNA vaccines. In order to assess a new strategy for oral DNA vaccine formulation and delivery, plasmid encoding hemagglutinin (HA) gene of avian influenza virus, A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1/H5) was formulated using green synthesis of sliver nanoparticles (AgNP) with polyethylene glycol (PEG). AgNP were successfully synthesized uniformly dispersed with size in the range of 4 to 18 nm with an average size of 11 nm. Cytotoxicity of the prepared AgNP was investigated in vitro and in vivo using MCF-7 cells and cytokine expression, respectively. At the concentration of − 5 log10AgNP, no cytotoxic effects were detected in MCF-7 cells with 9.5% cell death compared to the control. One-day-old specific pathogen-free (SPF) chicks immunized once by oral gavage with 10 μl of pcDNA3.1/H5 (200 ng/ml) nanoencapsulated with 40 μl AgNP (3.7 × 10− 2 μg of Ag) showed no clinical manifestations. PCR successfully detect the AgNP/H5 plasmid from the duodenum of the inoculated chicken as early as 1 h post‐immunization. Immunization of chickens with AgNP/H5 enhanced both pro inflammatory and Th1-like expressions, although no significant differences were recorded in the chickens inoculated with AgNP, AgNP/pcDNA3.1 and the control. In addition, serum samples collected from immunized chickens with AgNP/H5 showed rapidly increasing antibody against H5 on day 14 after immunization. The highest average antibody titres were detected on day 35 post‐immunization at 51.2 ± 7.5. AgNP/H5 also elicited both CD4+ and CD8+ T cells in the immunized chickens as early as day 14 after immunization, at 7.5 ± 2.0 and 20 ± 1.9 percentage, respectively. Hence, single oral administrations of AgNP/H5 led to induce both the antibody and cell-mediated immune responses as well as enhanced cytokine production