Preparation And Immune Efficacy Of Carbon Nanotubes Adjuvanted Recombinant Subunit Vaccine Against Aeromonas Hydrophila

Aeromonas hydrophila, a gram-negative bacterium, is widely distributed in freshwater and seawater. The bacterium can be an opportunistic pathogen in a variety of fish, amphibians, reptiles and humans, and regarded as one of the major problems in aquaculture. Of late, millions of dollars are estimated to be lost per annum due to the diseases caused by this bacterium. Presently, recombinant vaccine via intramuscular injection is considered as a prophylactic measure that has developed for many years. However, it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Therefore, bath immunization becomes an attractive option to prevent the disease. However, the efficacy of bath immunization is not ideal due to several barriers of skin epithelium. Hence, it is necessary to develop a novel immersion delivery vehicle to overcome these obstacles.Carbon nanotubes(CNTs), within the family of emerging nanomaterials, have very interesting characteristics of high drug loading. Functionalized CNTs seem to have a propensity to penetrate biological membranes and tissue barriers. These characteristics make CNTs as a unique material for transporting bioactive molecules such as drugs, vaccines, genes and other therapeutic agents. In this study, a recombinant aerA vaccine was generated through prokaryotic expression strategy, and a chemical modification method was utilized to link aerA and functionalized SWCNTs to prepare a novel SWCNTs-aerA vaccine, and the immune response on grass carp was evaluated in various ways. The results obtained in this work were as follows:1. The recombinant plasmid pET32a-aerA was successfully constructed. After induced by IPTG, a great amount of recombinant aerA protein(71.4 kDa) was obtained after the purification and renaturation procedures. Using the chemical modification methods, the aerA protein were linked to the surface of oxidized SWCNTs to prepare SWCNTs-aerA subunit vaccine system. Quantitative protein analysis showed that the percentage content of aerA was 41.6% in SWCNTs-aerA.2. Grass carps with the weight of 1.0 g were vaccinated by SWCNTs-aerA/aerA via intramuscular injection and bath immunization. The results showed that SWCNTs-aerA elicited higher levels of antibody compared with free aerA at the same immunization dose/concentration(P<0.05). Fish immunized by aerA at the highest dose(20 μg) or concentration(50 mg/L) obtained the relative percentage survival(RPS) of 48.1% and 47.4%, respectively. Meanwhile, SWCNTs-aerA immunized fish showed a much higher survival rate than the aerA, which correspond to RPS of 79.6% and 84.9%, respectively. The results suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach.3. To assess the effects that the SWCNTs-aerA vaccine may have on the gut microbiota, the study searched for differences in intestinal bacterial composition between vaccinated and unvaccinated fish. The results showed that the proportion of Aeromonas in SWCNTs-aerA /aerA immunized groups were 6.21% and 5.91%, respectively. However, the ratio changed to 6.92% and 4.85% after challenged by Aeromonas hydrophila. Meanwhile, the anundance in control group significantly increased from 10.43% to 19.48%. The results suggested that SWCNTs-aerA vaccine could maintain the Aeromonas in intestinal bacterial composition.This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate small fish by bath administration method. This work will also help us to further determine an efficient CNTs-vaccine delivery system in fish.