Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants

Abstract

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund's complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16-19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.

Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization

“…Similar to our previous study (Kubo et al, 2022a), the RV-N gene (accession number: AB069973) derived from a challengevirus-standard (CVS) strain of RV was ligated into a pcDNA 3.1 plasmid (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant plasmid DNA (pcDNA-N) was amplified in E. coli DH5α competent cells (Takara, Japan) and purified using the NucleoBond Xtra Midi plasmid DNA purification kit (Takara, Japan), according to the manufacturer's protocol.…”

“…Fig. 1 shows the immunization schedule used to immunize laying hens, as described previously (Kubo et al, 2022b). Fifteen hens (Boris Brown, 356 days old) were divided into five groups with three hens per group.…”

“…IgYs can be a promising substitute for purified Igs for treating rabies viral infections and can be used as a primary antibody in IHC (Motoi et al, 2005a;2005b). We also reported that RV nucleoprotein (RV-N)-specific antibodies can be prepared by DNA immunization combined 2 with various adjuvant injections (Kubo et al, 2022a). We further developed and reported an immunization method using intramuscular injection of an adjuvant before immunization of laying hens to increase antibody activity in DNA immunization (Kubo et al, 2022b).…”

“…If an IgY antibody could be prepared by DNA immunization for IHC detection, DNA immunization could be a simpler method for preparing antibodies for Ravies diagnosis. In this study, chicken IgY antibodies against RV-N prepared by both DNA immunization and conventional protein immunization into lying hens as described in our previous study (Kubo et al, 2022b) were used to investigate these characteristics by applying IHC against rabies-positive dog brain samples and performing enzyme-linked immunosorbent assay (ELISA) using undenatured or denatured RV-N as the coated antigen.…”

Novel extraction technologies and potential applications of egg yolk proteins

“…In addition, IgY have great potential applications in plants and animals’ health. For example, specific IgY against was reported to be effective in detecting wheat stripe mosaic virus (Santos et al, 2022 ), Furcraea Necrotic Streak Virus in fique plants (Toloza-Moreno et al, 2022 ), rabies viruses (Kubo et al, 2022 ), infectious bursal disease virus (Wibawan et al, 2018 ). IgY antibodies also exhibited protective effect against genotype XII Newcastle disease virus in chicken (Calderon et al, 2022 ), H9N2 avian influenza virus (Wang et al, 2020 ), duck adenovirus 3 (Yin et al, 2019 ), duck hepatitis B virus (Liu et al, 2018 ), etc.…”