Image_1_A therapeutic chimeric IgG/IgA expressed by CHO cells for oral treatment of PED in piglets.JPEG

Immunoglobulin A (IgA) of sows is critically important for assessing piglets’ protective capacity against porcine epidemic diarrhea virus (PEDV). Here, we report a therapeutic chimeric anti-PEDV IgG/IgA expressed by Chinese hamster ovary (CHO) cells for oral treatment of PED. The chimeric anti-PEDV IgG/IgA was produced by the CHO cell lines, in which the heavy chain was constructed by combining the VH, Cγ1 and hinge regions of PEDV IgG mAb 8A3, and the Cα2 and Cα3 domains of a Mus musculus immunoglobulin alpha chain. The chimeric anti-PEDV IgG/IgA could neutralize the strains of CV777 (G1), P014 (G2) and HN1303 (G2) in vitro effectively, showing broad-spectrum neutralization activity. The in vivo challenge experiments demonstrated that chimeric anti-PEDV IgG/IgA (9C4) produced in the CHO cell supernatant could alleviate clinical diarrhea symptoms of the PEDV infection in piglets. In general, our study showed that chimeric anti-PEDV IgG/IgA produced from CHO cell line supernatants effectively alleviates PEDV infection in piglets, which also gives the foundation for the construction of fully functional secretory IgA by the J chain introduction to maximize the antibody therapeutic effect.</p

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design

<div><p>Pseudorabies virus (PRV) belongs to the <i>Herpesviridae</i> family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.</p></div

Sequance characteristics of PRV neutralizing mAbs.

<p>Sequance characteristics of PRV neutralizing mAbs.</p

PRV neutralizing activity and protein ELISA reactivities of candidate antibodies.

<p>PRV neutralizing activity and protein ELISA reactivities of candidate antibodies.</p

Sequance characteristics of PRV neutralizing mAbs.

<p>Sequance characteristics of PRV neutralizing mAbs.</p

Neutralizing efficacies of 1H1 mAb against different PRV strains.

<p>Purified 1H1 mAb was used for virus neutralizing assay to test its efficacy against 8 different PRV strains, including vaccine strains (Bartha and HB98), classical virulent strains (RA and SU), and the current emerging variant strains (HN1201, 188–5, 072–1, and BH1). The neutralizing profile of each strain is represented by different symbols as indicated in the legend. Each experiment was conducted with 3 replicates. The error bars representing standard deviations from the mean are shown correspondingly.</p

Crystal structure of PRV gB and gB-D_IV.

<p>(A) Schematic representation of PRV gB architecture and the construct design of gB ectodomain and D_IV. Each domain is represented by different colors, and the positions of fusion loops and potential furin cleavage site are indicated by arrows. SP, signal peptide; MPR, most polymorphic region; TM, transmembrane region; Cyto, cytoplasmic tail; L67, signal peptide of gp67. (B) Overall structure of PRV gB trimer. One of the protomers is colored by domains as in (A) and labeled accordingly. The fusion loops are indicated by black arrows. (C) Superposition of PRV gB and HSV gB. The gB protomers and each individual domains are superimposed respectively. The rmsd values of Cα atom coordinates of aligned residues are shown under each comparison figure. (D) Crystal structure of PRV gB-D_IV, top and side views. The gB-D_IV exists as homotrimer and is colored by chains. (E) Structural alignment between the PRV gB trimer and D_IV.</p

Flow cytometry analysis to verify the 1H1 epitope by mutagenesis.

<p>The 293T cells were transfected by wild type (A) PRV gB or mutants (B-F) expression vectors to allow protein expression on the cell surface. The 1H1 mAb was then used to stain the transfected cells, which were further stained by APC-linked secondary antibody. The fluorescence signals of the cells were visualized and quantified by flow cytometry. The profiles of cells transfected with pEGFP-N1 empty vectors are represented by solid black areas (negative control) and the cells transfected with gB or mutant expression plasmids are represented by red silhouettes. WT, wild type; Mut4, quadruple mutants.</p