Mooring Stone-Like Arg114 Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 1*0101 (SLA-1*0101). We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg114 residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 1*0101 (SLA-1*0101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg114 of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.

Affinity-based isolation of extracellular vesicles and the effects on downstream molecular analysis.

Extracellular vesicles (EVs) are transport vesicles with diameters ranging from 30 to 1000 nm, secreted by cells in both physiological and pathological conditions. By using the EV shuttling system, biomolecular cargo such as proteins and genetic materials travels between cells resulting in intercellular communication and epigenetic regulation. Because the presence of EVs and cargo molecules in body fluids can predict the state of the parental cells, EV isolation techniques from complex biofluids have been developed. Further exploration of EVs through downstream molecular analysis depends heavily on those isolation technologies. Methodologies based either on physical separation or on affinity binding have been used to isolate EVs. Affinity-based methods for EV isolation are known to produce highly specific and efficient isolation results. However, so far, there is a lack of literature summarizing these methods and their effects on downstream EV molecular analysis. In the present work, we reviewed recent efforts on developing affinity-based methods for the isolation of EVs, with an emphasis on comparing their effects on downstream analysis of EV molecular cargo. Antibody-based isolation techniques produce highly pure EVs, but the harsh eluents damage the EV structure, and some antibodies stay bound to the EVs after elution. Aptamer-based methods use relatively mild elution conditions and release EVs in their native form, but their isolation efficiencies need to be improved. The membrane affinity-based method and other affinity-based methods based on the properties of the EV lipid bilayer also isolate intact EVs, but they can also result in contaminants. From the perspective of affinity-based methods, we investigated the influence of the isolation methods of choice on downstream EV molecular analysis.