Immunogenicity of a new, inactivated canine adenovirus type 2 vaccine for dogs

Abstract

Purpose: We constructed a new canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs.

Materials and methods: To generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA).

Results: The new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs.

Conclusion: The Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.

Keywords: Adjuvant; Canine adenovirus type 2; Vaccine.

Fig. 1. The APQA1701 strain (genome: 31,071 nucleotides) lacked two early proteins (encoded in the E1A region) and the pTP of the APQA1601 and Toronto A26/61 strains (A, B). The missing nucleotides are marked in red. APQA1701 grew to a titer of 10^6.5 TCID₅₀/mL in MDCK cells, but did not grow in Vero cells (C). pTP, precursor terminal protein.

Fig. 2. Genetic characterization of the APQA1701-40P variant generated over 40 passages using the sequential, limit dilution culture method. The entire genome was sequenced and compared to the reference CAV-2 sequences. APQA1701-40P exhibited an 18-nucleotide deletion in the E1b-19K gene (A); the protein thus lacked six amino acids (B). Polymerase chain reaction showed that APQA1701-40P variant arose after 14 passages (C). E312.5K amino acid sequences of the APQA1701 parent and APQA1701-40P. Amino acid 21 (alanine) of the parent gene was changed to glutamine (D). CAV-2, canine adenovirus type 2.

Fig. 3. Levels of serum IgG antibodies against CAV-2 in guinea pigs and dogs that received vaccines containing the IMS gel or Cabopol adjuvant. Dogs were immunized at 2-week intervals. Serum samples were collected at 0, 2, and 4 weeks post-vaccination and CAV-2-specific IgG levels measured in guinea pigs (A) and dogs (B). IgG, immunoglobulin G; CAV-2, canine adenovirus type 2.

Fig. 4. Titers of neutralizing antibodies against CAV-1 and CAV-2 in guinea pigs (A) and dogs (B) that received inactivated CAV-2 vaccines containing IMS gel or Cabopol. The titers against CAV-2 are higher than those against CAV-1 in both dogs and guinea pigs. CAV-1, canine adenovirus type 1; CAV-2, canine adenovirus type 2; VN, virus neutralization.