Comparison of Five Expression Vectors for the Ha Gene in Constructing a DNA Vaccine for H6N2 Influenza Virus in Chickens

Abstract

A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses; however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.

Evaluation of different chemical adjuvants on an avian influenza H6 DNA vaccine in chickens

“…In our previous report, the pCAG-HAk vaccine induced moderate antibody responses in SPF chickens (Shan et al, 2011), however, the same DNA vaccine did not induce measurable antibody in Hy-Line chickens (Shan et al, 2011). Furthermore, we found that even if other serological methods such as ELISA or western blot were used, no or very weak antibody responses were detected in the evaluation of the VR10102 vector vaccine expressing the same HA gene of H6N2 AIV (Shan et al, 2016). The reason for this is unknown but worth further investigation.…”

“…However, incorporation of either chemical or genetic adjuvants to DNA vaccines modulates or enhances their efficacy (Cui & Mumper, 2003a). As our previous study demonstrated, lipofectin (liposome) was able to generate an enhanced DNA-induced immune response (Shan et al, 2016). In the present study, aluminum hydroxide (traditional adjuvant), Essai (microparticle) and Phema (nanoparticle) were assessed to see if the humoral immune response could be enhanced.…”

“…Thereafter DNA vaccination against avian influenza has been widely studied in chickens, but their capacity to induce antibody responses has been shown to be variable in chickens (Kodihalli et al, 1997;Suarez & Schultz-Cherry, 2000;Le Gall-Recule, et al, 2007;Shan et al, 2011;Jazayeri, et al, 2012;Ogunremi, et al, 2013;Meunier, et al, 2016;Shan et al, 2016). Moreover, promising results have been mainly obtained in SPF birds (Meunier, et al, 2016).…”

“…In a previous experiment (Shan et al, 2016), the chemical adjuvant lipofectin showed some enhancement of an avian influenza HA DNA-induced immune response in chickens in terms of increasing the seroconversion rate of vaccinated birds and the level of antibody production, and reducing virus shedding in both oropharyngeal and cloacal swabs. However, it is prohibitively expensive for routine use in poultry.…”

“…The pCAG-HAk plasmid DNA expressing the complete haemagglutinin (HA) gene of avian influenza A/Eurasian coot/Western Australia/2727/1979 (H6N2) virus, together with a Kozak sequence, in a pCAGGS vector, was used as a DNA vaccine as described previously (Shan et al, 2011). The optimized DNA dose had been determined previously for this plasmid and for four other expression vectors (Shan et al, 2011;Shan et al, 2016). Preparation of DNA-coated alum.…”