Table 1.
Age, gender, ABO group, dietary and vaccination histories, and geographic area of childhood in 84 healthy human subjects from whom sera were studied in the present report.
Fig 1.
Expression of Gal, Neu5Gc, and hCD46 on wild-type, GTKO/CD46, GTKO/CD46/Neu5GcKO pig and human RBCs and AECs by flow cytometry.
RBCs and AECs from GTKO/CD46 pigs expressed Neu5Gc, but not Gal. AECs from these pigs expressed hCD46, but RBCs did not. Neither Gal nor Neu5Gc was expressed on GTKO/CD46/Neu5GcKO pig RBCs or AECs. Human AECs expressed hCD46, but not Gal or Neu5Gc.
Fig 2.
Definitions of anti-pig IgM and IgG antibodies.
Anti-pig antibodies (both IgM and IgG) such as anti-Gal, anti-Neu5Gc, anti-nonGal, and anti-nonGal/nonNeu5Gc were defined according to binding to various pig cells.
Fig 3.
[A] Human serum IgM/IgG binding to GTKO/CD46 and GTKO/CD46/Neu5GcKO pRBCs and pAECs (flow cytometry). There was no significant difference in IgM or IgG binding to GTKO/CD46 pRBCs or pAECs (P>0.05). IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs were both significantly less than to the equivalent pAECs (P<0.01) although the levels of binding to pAECs were lower. [B] Difference between IgM/IgG binding to GTKO/CD46 and GTKO/CD46/Neu5GcKO pRBCs. There was a significant decrease in human IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs when compared with binding to GTKO/CD46 pRBCs (P<0.01). IgM binding decreased in 75% of sera (63/84), though a very small number (3.6%, 3/84) were found to have very slightly increased (statistically insignificant) binding; the relative GM decreased from >8 to <1. IgG binding decreased in 65% of sera (55/84), and increased minimally in 2.4% (2/84); the relative GM was reduced from >5 to <1. [C] Difference between IgM/IgG binding to GTKO/CD46 and GTKO/CD46/Neu5GcKO pAECs. There was significantly less IgM and IgG binding to cells from GTKO/CD46/Neu5GcKO pAECs than to GTKO/CD46 pAECs (p<0.01). IgM binding was reduced in 100% of sera, and IgG in 93% of sera (78/84). Relative GM fell from 4 to 2 (IgM) and from 2 to 1.5 (IgG). No sera showed an increase in IgG binding to GTKO/CD46/Neu5GcKO cells, but 7.2% (6/84) showed no change in binding.
Table 2.
Incidence (and percentage) of human serum IgM and IgG antibodies binding to pRBCs and pAECs.
Fig 4.
[A] Human IgM/IgG binding to Gal and Neu5Gc (ELISA). The level of anti-Gal IgM/IgG antibodies were higher than those of anti-Neu5Gc antibodies (P<0.0001). The level of anti-Gal and Neu5Gc IgG was significantly higher than that of anti-Gal and anti-Neu5Gc IgM (P<0.0001). [B] Correlation between human serum anti-Gal and anti-Neu5Gc IgM and IgG antibody levels. There is no correlation between anti-Gal level and anti-Neu5Gc level both in IgM and IgG (P>0.05). [C] Correlation of human serum anti-Neu5Gc IgM and IgG antibody with age (ELISA). There was no correlation between either anti-Neu5Gc IgM or IgG levels with age (P>0.05). [D] Difference in human anti-Neu5Gc antibody between gender (ELISA). There was no significant difference in anti-Neu5Gc IgM levels between female and male (P>0.05), but the level of anti-Neu5Gc IgG in males was significantly higher than in females (P<0.05).
Table 3.
Correlation of human serum anti-Neu5Gc IgM and IgG antibodies with vaccination history.
Fig 5.
[A] Correlation between anti-nonGal and anti-nonGal/nonNeu5Gc IgM and IgG antibody levels. When pRBCs were the target cells, there was no correlation between anti-nonGal IgM and anti-nonGal/nonNeu5Gc IgM (P>0.05). There was significant positive correlation between anti-nonGal IgG and anti-nonGal/nonNeu5Gc IgG (P<0.05). When pAECs were the target cells, there was highly significant positive correlation between anti-nonGal and anti-nonGal/nonNeu5Gc IgM/IgG (P<0.001). [B] Correlation between anti-Neu5Gc, anti-nonGal, and anti-nonGal/nonNeu5Gc IgM. When pRBCs were the target cells, there was a significant negative correlation between anti-Neu5Gc IgM and anti-nonGal/nonNeu5Gc IgM (P<0.05), but no correlation between anti-Neu5Gc IgM and anti-nonGal IgM. When pAECs were the target cells, there was no correlation between anti-nonGal IgM and anti-nonGal/nonNeu5Gc IgM (P>0.05). [C] Correlation between anti-Neu5Gc, anti-nonGal, and anti-nonGal/nonNeu5Gc IgG. When pRBCs were the target cells, there was a highly significant positive correlation between anti-Neu5Gc IgG and anti-nonGal IgG (P<0.001). When pAECs were the target cells, there was a significant positive correlation between anti-Neu5Gc IgG and anti-nonGal IgG (P<0.05). There is no correlation between anti-Neu5Gc IgG and anti-nonGal/nonNeu5Gc IgG both to pRBCs and pAECs (P>0.05).