Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited

Abstract

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise ∼80% of the preimmune repertoire. However, so-called “hybrid” VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied >3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for >95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.

Antibody Repertoire Development in Fetal and Neonatal Piglets: XIX. Undiversified B Cells with Hydrophobic HCDR3s Preferentially Proliferate in the Porcine Reproductive and Respiratory Syndrome

“…The deduced amino acid sequence of the various CDR3 regions extending from, but not including, the FR3 cysteine at position 104 down to the 5Ј region of J H , but not including the invariant tryptophan that starts FR4, were analyzed as described by Kyte and Doolittle (62) using the principles described by Eisenberg (63) as applied to HCDR3 sequences by Ivanov et al (64). HCDR3 sequences recovered from C␥ transcripts from PRRSV-infected piglets were compared with those from sham controls, SIV-infected piglets, colonized isolator piglets, and conventionally reared parasite-infected young pigs (PIC) (50,61). Sequence data are based on Ͼ415 transcript, 60 -100 for each treatment group.…”

“…Following secondary amplification the resulting product was cloned into pCR2 TOPO (Invitrogen Life Technologies). V H gene usage was determined by clonal hybridization using probes specific for the CDR1 and CDR2 region of the major porcine V H gene (58,61,65).…”

“…V H gene usage by IgM and IgG transcripts from PRRSV-infected piglets and sham controls was determined as described elsewhere (58,61,65). Briefly, rearranged VDJs transcribed with IgM and IgG were recovered from cDNA by PCR.…”

“…These were processed as previously described for the preparation of total RNA and subsequently cDNA (12,60). VDJs expressed on IgM and IgG transcripts were recovered as described previously (60,61). HCDR3 regions for spectratypic analysis of Ig were amplified using a FR3-J H primer set as described previously (50).…”

Antibody Repertoire Development in Fetal and Neonatal Piglets. XX. B Cell Lymphogenesis Is Absent in the Ileal Peyer’s Patches, Their Repertoire Development Is Antigen Dependent, and They Are Not Required for B Cell Maintenance

“…However, swine have multiple offspring (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), weigh ,2 kg at birth, and can be conveniently reared in GF isolators after their recovery by cesarean surgery where all environmental and maternal factors are thereafter controlled by the experimenter (41,42). Furthermore, the V H repertoire of swine is well characterized, partially mapped, and has been followed during development using methods that quantify repertoire development and frequency of SHM (43)(44)(45)(46)(47).…”