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

Abstract

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.

Keywords: DNA immunization; FCA; IHC; IgY; Rabies virus; λ-carrageenan.

Fig. 1.

Immunization schedule. FCA: Freund’s complete adjuvant; FIA: Freund’s incomplete adjuvant; W: week.

Fig. 2.

Comparison of IgY ELISA values (absorbance at 405 nm) in egg yolk. The results of the ELISA performed using egg yolk collected at the 8th week are shown. ELISA was performed for 10 min at 37°C. Egg yolks from individual hens (indicated by *) were used for IgY purification. Group 1, control; Groups 2 and 3, DNA immunization (with pre-immune stimulation) using λ-carrageenan or Freund’s complete adjuvant, respectively; Groups 4 and 5, conventional protein immunization with rRV-N in 0 M Uurea or rRV-N in 4 M Urea, respectively.

Fig. 3.

Reactivities of purified IgYs against RV-N vaccine (ELISA). The reactivity of each purified IgY was demonstrated when the rabies virus vaccine was used as the solid-phase antigen. Data represent mean ± SD (n = 3). Different lowercase letters above bars indicate statistically significant differences (P < 0.05) for each IgY dilution.

Fig. 4.

Immunohistochemical (IHC) staining. Brain sections (hippocampus, formalin-treated) of rabies-infected dogs were used as samples for IHC and observed under an optical microscope (400×). Rabies virus inclusion bodies (Negri bodies) are stained red in IV and V (arrowheads).

Fig. 5.

Evaluation of binding activity between denatured antigen and various IgY antibodies in ELISA. Reactivities between various purified anti-RV-N IgY antibodies and untreated (I); 10% formalin-treated (II); 60°C, 30 min heat-treated after formalin treatment (III); and 60°C, 30 min and 90°C, 5 min heat-treated after formalin treatment (IV) antigens measured by ELISA. The horizontal axis shows the dilution ratio of the solid-phase antigen [10 (F10), 100 (F100), 1000 (F1000) times dilution from the vaccine stock solution]. Data represent mean ± SD (n = 3). Different lowercase letters above bars indicate statistically significant differences (P < 0.05) for each antigen dilution ratio.