A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2

Abstract

A recent outbreak of hepatitis-hydropericardium syndrome caused by serotype 4 fowl adenovirus (FAdV-4) has resulted in significant economic losses to the poultry industry worldwide. However, little is known about the molecular pathogenesis of FAdV-4. In this study, a novel monoclonal antibody (mAb) targeting the fiber-2 protein of FAdV-4 was generated, mAb 3C2. Indirect immunofluorescence assay showed that mAb 3C2 neither reacted with serotype 8 fowl adenovirus (FAdV-8) nor reacted with the fiber-1 protein of FAdV-4; it specifically reacted with the fiber-2 protein of FAdV-4. Notably, mAb 3C2 could efficiently immunoprecipitate the fiber-2 protein in chicken liver cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2. Moreover, mAb 3C2 demonstrated marked neutralizing activity against FAdV-4 and could efficiently inhibit the infection of FAdV-4 in vitro. Using truncated fiber-2 constructs, the epitope recognized by mAb 3C2 was determined to be located between amino acids 416-448 at the C-terminus of fiber-2. Our data not only provide a foundation for the establishment of a rapid fiber-2 peptide-based diagnostic assay for FAdV-4 but also highlight the critical role of the fiber-2 protein in mediating infection by FAdV-4. Furthermore, the epitope recognized by 3C2 might serve as a novel target for the development of a vaccine targeting FAdV-4.

Figure 1

SDS-PAGE analysis of the purified 3C2. 3C2 ascites were purified with protein G and analysed by SDS-PAGE. Lane M: Protein Marker Blue Plus II; lane 1: purified 3C2; lane 2: non-purified 3C2.

Figure 2

Specificity analysis for 3C2 by IFA. LMH cells were infected with either FAdV-4 or FAdV-8 or transfected with pcDNA3.1-F1 or pcDNA3.1-F2. Then, 3C2 was used as the primary antibody to perform IFA to analyse its specificity. A, B 3C2 reacted with chicken liver cells infected with FAdV-4 but not FAdV-8. C, D 3C2 reacted with LMH cells transfected with pcDNA3.1-F2 but not pcDNA3.1-F1.

Figure 3

Immunoprecipitation analysis. LMH cells were infected with FAdV-4 or transfected with pcDNA3.1-F2. Then, 3C2 was used as the capture antibody to perform immunoprecipitation, followed by Western blot analysis using chicken sera against FAdV-4. A Lanes 1 and 2: lysates of LMH cells infected with or without FAdV-4, respectively. Lanes 3 and 4: 3C2-immunoprecipitated pellets of LMH cells infected with or without FAdV-4, respectively. B Lanes 1 and 2: lysates of LMH cells transfected without or with pcDNA3.1-F2, respectively. Lanes 3 and 4: 3C2-immunoprecipitated pellets of LMH cells transfected without or with pcDNA3.1-F2, respectively.

Figure 4

IFA analysis for the neutralization activity of 3C2. Serial dilutions of 3C2 or control 6E6 mAbs were first mixed with FAdV-4, and then, LMH cells were infected with the mixture and analysed by IFA, as described in “Materials and methods”. A–D and I–L LMH cells infected with the mixture of virus and serial dilutions of 3C2; E–H and M–P LMH cells infected with the mixture of virus and serial dilutions of mAb 6E6.

Figure 5

Western blot analysis of the neutralization activity of 3C2. 1:200 and 1:5000 dilutions of 3C2 or control 6E6 mAbs were first mixed with FAdV-4, and then LMH cells were infected with the mixture and analysed by Western blot using chicken sera against FAdV-4, as described in “Materials and methods”. Lanes 1 and 2: lysates of LMH cells infected with the mixture of virus and the 1:200 dilutions of 3C2 and 6E6, respectively. Lanes 3 and 4: lysates of LMH cells infected with or without FAdV-4, respectively. Lanes 5 and 6: lysates of LMH cells infected with the mixture of virus and 1:5000 dilutions of 3C2 and 6E6, respectively.

Figure 6

Epitope mapping for mAb 3C2 using truncated fiber-2 constructs. LMH cells transfected with the different truncated fiber-2 constructs respectively were fixed, and stained with mAb 3C2 or chicken sera against FAdV-4 against FAdV-4 through IFA. A–I The LMH cells were transfected with pcDNA3.1-F2, F2_1-415aa, F2_1-382aa, F2_1-349aa, F2_1-316aa, F2_1-283aa, F2_1-250aa, F2_1-217aa and F2_1-182aa respectively.