No abstract

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease that inflicts huge economic damage on the swine industry worldwide. A safe and highly effective PEDV vaccine that contains only the virus-neutralizing epitopes (not enhancing epitope), as well as a ready-to-use PEDV neutralizing antibody for the passive immunization of PEDV vulnerable piglets (during the first week of life) are needed, particularly for PEDV-endemic farms. In this study, we generated monoclonal antibodies (mAbs) to the recombinant S1 domain of PEDV spike (S) protein and tested their PEDV neutralizing activity by CPE-reduction assay. The mAb secreted by one hybrodoma clone (A3), that also bound to the native S1 counterpart from PEDV-infected cells (tested by combined co-immunoprecipitation and Western blotting), neutralized PEDV infectivity. Epitope of the neutralizing mAb (mAbA3) locates in the S1A subdomain of the spike protein, as identified by phage mimotope search and multiple sequence alignment, and peptide binding-ELISA. The newly identified epitope is shared by PEDV G1 and G2 strains and other alphacoronaviruses. In summary, mAbA3 may be useful as a ready-to-use antibody for passive immunization of PEDV-susceptible piglets, while the novel neutralizing epitope, together with other, previously known protective epitopes, have potential as an immunogenic cocktail for a safe, next-generation PEDV vaccine.

Coimplantation of endothelial cells (ECs) and mesenchymal stromal cells (MSCs) into the transplantation site could be a feasible option to achieve a sufficient level of graft-host vascularization. To find a suitable source of tissue that provides a large number of high-quality ECs and MSCs suited for future clinical application, we developed a simplified xeno-free strategy for isolation of human umbilical vein endothelial cells (HUVECs) and Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) from the same umbilical cord. We also assessed whether the coculture of HUVECs and WJ-MSCs derived from the same umbilical cord (autogenic cell source) or from different umbilical cords (allogenic cell sources) had an impact on in vitro angiogenic capacity. We found that HUVECs grown in 5 ng/ml epidermal growth factor (EGF) supplemented xeno-free condition showed higher proliferation potential compared to other conditions. HUVECs and WJ-MSCs obtained from this technic show an endothelial lineage (CD31 and von Willebrand factor) and MSC (CD73, CD90, and CD105) immunophenotype characteristic with high purity, respectively. It was also found that only the coculture of HUVEC/WJ-MSC, but not HUVEC or WJ-MSC mono-culture, provides a positive effect on vessel-like structure (VLS) formation, in vitro. Further investigations are needed to clarify the pros and cons of using autogenic or allogenic source of EC/MSC in tissue engineering applications. To the best of our knowledge, this study offers a simple, but reliable, xeno-free strategy to establish ECs and MSCs from the same umbilical cord, a new opportunity to facilitate the development of personal cell-based therapy.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80–100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.