Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.

Antibody upstream sequence diversity and its biological implications revealed by repertoire sequencing

The sequence upstream of the antibody variable region(antibody upstream sequence[AUS])consists of a 5′untranslated region(5′UTR)and a preceding leader region.The sequence variations in AUS affect antibody engineering and PCR based antibody quantification and may also be implicated in mRNA transcription and translation.However,the diversity of AUSs remains elusive.Using 5′rapid amplification of cDNA ends and high-throughput antibody repertoire sequencing technique,we acquired full-length AUSs for human,rhesus macaque,cynomolgus macaque,mouse,and rat.We designed a bioinformatics pipeline and identified 3307 unique AUSs,corresponding to 3026 and 1457 unique sequences for 5′UTR and leader region,respectively.Comparative analysis indicated that 928(63.69%)leader sequences are novel relative to those recorded in the international ImMunoGeneTics information system.Evolutionarily,leader sequences are more conserved than 5′UTR and seem to coevolve with their downstream V genes.Besides,single-nucleotide polymorphisms are position dependent for leader regions and may contribute to the functional reversal of the downstream V genes.Finally,the AUGs in AUSs were found to have little impact on gene expression.Taken together,our findings can facilitate primer design for capturing antibodies efficiently and provide a valuable resource for antibody engineering and molecule-level antibody studies.

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection

Advancements in high-throughput sequencing(HTS)of antibody repertoires(Ig-Seq)have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale.However,currently,only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions,possibly limited by inadequate sequencing depth and throughput.To better understand how HIV-1 infection would impact humoral immune system,in this study,we systematically analyzed the differences between the IgM(HIV-IgM)and IgG(HIV-IgG)heavy chain repertoires of HIV-1 infected patients,as well as between antibody repertoires of HIV-1 patients and healthy donors(HH).Notably,the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries,and the diversity of unique clones in HIV-IgG remarkably reduced.In aspect of somatic mutation rates of CDR1 and CDR2,the HIV-IgG repertoire was higher than HIV-IgM.Besides,the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire,presumably caused by the great number of novel VDJ rearrangement patterns,especially a massive use of IGHJ6.Moreover,some of the B cell clonotypes had numerous clones,and somatic variants were detected within the clonotype lineage in HIV-IgG,indicating HIV-1 neutralizing activities.The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.