Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.
ABSTRACT H5N1 influenza viruses have caused significant disease and deaths in various parts of the world in several species, including humans. Vaccination combined with culling can provide an attractive method for outbreak containment. Using synthesized oligos and overlapping extension PCR techniques, we constructed an H5 HA gene, optiHA, containing chicken biased codons based on the HA amino acid sequence of the highly pathogenic H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The optiHA and wild-type HA genes were inserted into plasmids pCI or pCAGGS, and designated as pCIoptiHA, pCAGGoptiHA, pCIHA and pCAGGHA, respectively. To evaluate vaccine efficacy, groups of 3-week-old specific pathogen free (SPF) chickens were intramuscularly injected with the four plasmids. Sera were collected on a weekly basis post-vaccination (p.v.) for hemagglutination inhibition (HI) assays and neutralization (NT) antibody detection. All chickens receiving pCAGGoptiHA and pCAGGHA developed high levels of HI and NT antibodies at 3 weeks p.v., and were completely protected from lethal H5 virus challenge, while only partial protection was induced by inoculation with the other two plasmids. A second experiment was conducted to evaluate if a lower dose of the pCAGGoptiHA vaccine could be effective, results indicated that two doses of 10 microg of pCAGGoptiHA could induce complete protection in chickens against H5 lethal virus challenge. Based on our results, we conclude that construction optimization could dramatically increase the H5 HA gene DNA vaccine efficacy in chickens, and therefore, greatly decrease the dose necessary for inducing complete protection in chickens.
- SourceAvailable from: Róża Sawicka
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- "In other works a very broad range of doses was tested and showed to be effective. Usually around 10–200 lg ensured high or average level of response and protection       . "
ABSTRACT: Highly pathogenic avian influenza viruses (HPAIVs) cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA), are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments) indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated) transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8 weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus.01/2014; 3:40–46. DOI:10.1016/j.trivac.2014.02.002
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- "Avian influenza virus (AIV) contain a major surface glycoprotein , hemagglutinin (HA), which primarily involved in induction of specific humoral immunity and protective immune response against influenza virus (Jiang et al., 2007). However, the HA gene is extremely variable in sequence with 16 defined subtypes where antibody to one subtype will neutralize only virus in the same subtype (Sylte et al., 2007). "
ABSTRACT: We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.Research in Veterinary Science 08/2013; DOI:10.1016/j.rvsc.2013.07.013 · 1.51 Impact Factor
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- "Based on our results, we conclude that construction optimization could dramatically increase the H5 HA gene DNA vaccine efficacy in chickens, and therefore, greatly decrease the dose necessary for inducing complete protection in chickens (Jiang et al., 2007). Improvement of hemagglutinin (HA) expression of influenza virus has been optimized and synthesized the whole length of HA gene of H5N1 in accordance with the human's codon and inserted it to the eukaryotic expression vector pDC315 to construct a eukaryotic expression vector shows the better expression than wild type of gene (Li et al., 2008). "
ABSTRACT: A large number of influenza A virus outbreaks and mortality occurred in the world recently, an urgent attention to develop effective and sufficient quantity of vaccines are needed. Vaccines are generally protein with immunogenic properties and are not expressed in sufficient quantity because of the codon bias, so it is necessary to optimize its codon in the expression host. Codon optimization was used to improve the protein expression in living organisms by increasing the translational efficiency of gene of interest. Two surface antigenic glycoproteins, hemagglutinin (HA) and neuraminidase (NA) are present in influenza A viruses. We have used HA and NA genes from 19 strains of influenza A viruses for codon optimization in E. coli. Both genes of the influenza virus show that the codon adaptation index (CAI) and GC content of the genes in optimized DNA were enhanced significantly (p <0.01) as compared to wild type. CAI and GC of HA in optimized DNA was enhanced by 3.2 (68.5%) and 1.2 (16.2%) fold respectively, while in NA it was increased by 3.3 (69.7%) and 1.2 (15.8%) fold respectively. Our finding demonstrates that the optimized genes could be useful for better expression in host without any truncated proteins and also helpful for protein folding and function. This work provides new insight in the synthetic biology research.Interdisciplinary Sciences Computational Life Sciences 03/2011; 3(1):36-42. DOI:10.1007/s12539-011-0055-z · 0.66 Impact Factor