Carboxymethyl chitosan bounded iron oxide nanoparticles and gamma‐irradiated avian influenza subtype H9N2 vaccine to development of immunity on mouse and chicken

BACKGROUND: Avian influenza virus (AIV) subtype H9N2 is a low pathogenic avian influenza virus (LPAIV). OBJECTIVE: This study aims to evaluate the humoral and cellular immunity in vaccinated mice and broiler chicken by irradiated AIV antigen plus carboxymethyl chitosan bounded iron oxide nanoparticles (CMC‐IO NPs) as an adjuvant. METHODS: AIV subtype H9N2 with 10(8.5) EID(50)/ml and haemagglutinin antigen assay about 10 log(2) was irradiated by 30 kGy gamma radiation dose. Then, the gamma‐irradiated AIV was used as an inactivated vaccine and conjugated with CMC‐IO NPs to improve immune responses on mice. IO NPs must be applied in all activated tests using 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) and N‐hydroxysulfosuccinimide sodium salt (sulfo‐NHS), and then functionalized by CMC as IO‐CMC. Fourier transform infrared (FTIR) spectra on functionalized IO‐CMC showed a peak of 638 cm(−1) which is a band between metal and O (Fe‐O). RESULTS: Based on the comparison between the two X‐ray diffraction (XRD) patterns on Fe(2)O(3)‐NPs and IO‐CMC, the characteristics of IO‐NPs did not change after carboxymethylation. A CHN Analyzer was applied to measure the molecular weight of IO‐CMC that was calculated as 1045 g. IO‐CMC, irradiated AIV‐IO‐CMC and formalin AIV‐IO‐CMC were injected into 42 BALB/c mice in six groups. The fourth group was the negative control, and the fifth and sixth groups were inoculated by irradiated AIV‐ISA70 and formalin AIV‐ISA70 vaccines. An increase in haemagglutination inhibition (HI) antibody titration was observed in the irradiated AIV‐IO‐CMC and formalin AIV‐IO‐CMC groups (p < 0.05). In addition, increases in the lymphoproliferative activity of re‐stimulated splenic lymphocytes, interfron‐γ (IFN‐γ) and interleukin‐2 (IL‐2) concentration in the irradiated AIV‐IO‐CMC group demonstrated the activation of Type 1 helper cells. The concentration of IL‐4 was without any significant increases in non‐group. CONCLUSIONS: Accordingly, Th2 activation represented no increase. Finally, the finding showed that AIV‐IO‐CMC was effective on enhancing immunogenicity as irradiated AIV antigen administered with a clinically acceptable adjuvant (i.e. IO‐CMC).