Characterization of mAbs to chicken anemia virus and epitope mapping on its viral protein, VP1

Abstract

Three (MoCAV/F2, MoCAV/F8 and MoCAV/F11) of four mouse mAbs established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at~50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50 kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize five of the strains, indicating distinct antigenic variation amongst the strains. In blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/ F8 to the antigens and vice versa, suggesting that the two mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T) and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least two different neutralizing epitopes. However, MoCAV/F8 did not react with EsCAV/F2 or EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/ F2, with a titre of 1 : 12 800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titres of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.

Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus

“…First, the antigenicity of the CAV strain that infected the breeder chickens could have been different from that of the vaccine strain; however, the reactivity of the CAV strain (HK1/13) isolated from the diseased chicks was not different from the polyclonal antibody raised against the A2/76 strain (data not shown), and amino acid properties of the strain (accession no. KJ126838) were comparable with those of the other reported strains (Trinh et al, 2015). It has been reported that antigenic differences were not observed among CAV isolates using chicken polyclonal antibodies Yuasa and Imai, 1986).…”

“…A neutralizing mAb (MoCAV/F11, IgG2b) generated by our research group (Trinh et al, 2015) was used in the current study. Ascites fluid containing MoCAV/F11 was obtained following the intraperitoneal injection of approximately 10 7 hybridoma cells into a BALB/c mouse that had been primed with incomplete Freund's adjuvant, as described previously (Harlow and Lane, 1988).…”

Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-γ

“…It is widely accepted that neutralizing antibodies could possibly represent a valuable parameter by which to evaluate the efficacy of a vaccine against CAV (Sawant et al, 2015;Trinh et al, 2015b). Induction of cellular immune responses of IFN-␥ has been reported for a variety of antigens in previous studies (Haq et al, 2011;Takehara et al, 2003;van Slooten et al, 2000). Likewise, cell-mediated immunity, particularly the level of virusspecific IFN-␥, has been another potential correlate of protective immunity (Markowski-Grimsrud and Schat, 2003;Moeini et al, 2011;Sawant et al, 2015).…”

“…2a shows that rPiIFN-␥ can effectively enhance humoral immune responses. It is widely accepted that neutralizing antibodies could possibly represent a valuable parameter by which to evaluate the efficacy of a vaccine against CAV (Sawant et al, 2015;Trinh et al, 2015b). Induction of cellular immune responses of IFN-␥ has been reported for a variety of antigens in previous studies (Haq et al, 2011;Takehara et al, 2003;van Slooten et al, 2000).…”

“…vaccines have limitations including the inability of the virus to grow to high titers in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence (Sawant et al, 2015). Many studies have shown that the structural protein VP1 carries critical epitopes responsible for the induction of neutralizing antibodies (Trinh et al, 2015a). VP1 is thought to be a good candidate for use as an antigen for subunit vaccine development (Koch et al, 1995).…”