Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice.

The protective immune response against porcine circovirus 2 (PCV2) infection in mice was characterized using flow cytometric analysis (FCM), assays of antibody (of different IgG isotypes) and viraemia, and histopathological examination. An open reading frame 2 plasmid (pORF2) and the capsid protein (Cap) of PCV2 were used as DNA and subunit vaccines, respectively. In FCM analysis, although pORF2 and Cap alone showed comparable efficacy in eliciting lymphoproliferative responses and Cap-specific CD4(+) T cells, pORF2 was superior to the Cap protein in triggering CD8(+) T cells. A virus neutralization assay showed that pORF2 evoked stronger recall virus-neutralizing (VN) antibody responses than the Cap protein on PCV2 challenge. Correspondingly, VN antibody kinetics coincided with those of Cap-specific IgG2a, but not with the kinetics of IgG and IgG1. Following virus challenge, real-time PCR and histopathological analysis confirmed that only low viral DNA loads and mild microscopic lesions appeared in pORF2-immunized mice. These findings indicate that CD8(+) T cells and VN antibody responses correlating mainly with Cap-specific IgG2a play crucial roles in protecting against PCV2 infection, and that the protective immunity induced by the pORF2 plasmid is superior to that induced by the PCV2 Cap protein.

Development and validation of a recombinant capsid protein-based ELISA for detection of antibody to porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) has been recently associated with a number of disease syndromes, especially postweaning multisystemic wasting disease (PMWS). Herein, an alternative indirect enzyme-linked immunosorbent assay (ELISA) for detection of PCV2 antibody was developed using nuclear localization signal-truncated capsid protein of PCV2 produced in Escherichia coli (CAP ELISA). This assay was validated by comparison with an indirect immunofluorescence assay (IIF) and a PCV2-based ELISA. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the CAP ELISA were 95.3%, 93.9% and 95.1%, compared with IIF on 1080 field serum samples, and 93.3%, 84.2% and 91.1%, compared with the PCV2-based ELISA on 79 field sera, respectively. Cross-reactivity assay showed that this assay was PCV2-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 15%. This ELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PCV2 infection at low cost and the evaluation of the efficiency of various vaccines against PCV2.

Characterization of chicken interleukin 2 receptor alpha chain, a homolog to mammalian CD25.

To identify chicken IL-2R alpha chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL-2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease.

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal.

We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

Cloning, in vitro expression and bioactivity of duck interleukin-2.

In this report, the cDNA sequences of Shaoxing (SX) and Muscovy (MV) duck IL-2 were cloned, then recombinant duck IL-2 (rduIL-2) was produced in prokaryotic expression system. In vitro bioactivity of rduIL-2 was determined by lymphocyte proliferation assay and in vivo bioactivity of rduIL-2 was assessed by vaccine immunization. Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) specific for rduIL-2 were generated and characterized by ELISA, Western blot and neutralizing assays. The cDNA contains an open reading frame (ORF) of 420-base pairs encoding a protein of 140 amino acids (aa) with a putative signal peptide of 21aa. The His-duIL-2 fusion protein was recognized in Western blot by mAb against chicken IL-2 (chIL-2), but not by mAbs against human IL-2 and mouse IL-2. Recombinant duIL-2 induces in vitro proliferation of Con A-stimulated duck splenocytes in MTT assay and strengthens duck immune responses induced by vaccinating the inactivated oil emulsion vaccine against avian influenza virus. Polyclonal antibodies and mAb 2B3 against rduIL-2 were shown to have effective neutralizing ability by inhibiting the biological activities of both recombinant duIL-2 and endogenous duIL-2. Despite the fact that duck and chicken IL-2s only share identity of 55.0-56.7% in amino acid sequence, duck and chicken IL-2 molecules displayed similar cross-priming activity in in vitro lymphocyte proliferation assays. The results, at the first time, indicated that rduIL-2 has the potential to be used as an immunoadjuvant for enhancing vaccine efficacy and an immunotherapeutic, and the mAbs against rduIL-2 further facilitate basic immunobiological studies of the role of IL-2 in avian immune system.

A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function.

We combined the specificity of tumor-specific antibody with the chemokine function of interferon-gamma inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V(H) region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli.

A functional single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was produced at high level in Escherichia coli. The variable regions of heavy chain (V(H)) and light chain (V(L)) from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction. The construct of V(H)-linker-V(L) was inserted into a phagemid pCANTAB 5 E followed by selection with the Recombinant Phage Antibody System (RPAS). Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75% confirmed by spectrophotometer. The activity of refolded scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed anti-AIF scFv retains the specific binding activity to AIF with an affinity constant of 7.29 x 10(-8) mol l(-1). The overall yield of anti-AIF scFv with bioactivity in E. coli flask culture was more than 60 mg l(-1).