P10.14 LB
Background: Bovine immunoglobulins (Ig) typically have variable third heavy complementarity determining regions (CDRH3) for antigen engagement that are significantly longer than in humans and other mammals. We aimed to isolate HIV-1 bovine memory B cells binding HIV-1AD8 Env gp140 trimer antigen from a vaccinated cow producing broadly neutralising antibodies to examine the structure of their HIV Env gp140 antigen binding sites and construct chimeric bovine-human antibodies.
Methods: A cow was vaccinated with HIV-1AD8 Env gp140 trimers over 4 years and antigen-specific memory B-cells. HIV specific memory B cells were detected in ELISPOT assay and isolated by FACS single-cell sorting from PBMC. The bovine Ig heavy (H) and light (L) chain variable (V) regions were amplified from cDNA from anti-CD21+ anti-IgG+ gp140-PE-binding+ cells by nested PCR. Paired H and L chain expression vectors using human Ig constant regions were made and co-transfected into 293T cells. Chimeric bovine-human (BH) Ig in supernatant was screened for HIV-1 gp140 binding in ELISA.
Results: The frequency of HIV specific memory B cells was 1.96% ± 0.31% of total memory B cells on a background of 0.24% in non-immune PBMC. Ig from 6 of 30 matched chimeric BH H and L chain plasmid transfections displayed strong binding to HIV-1 AD8 gp140 Env trimers using direct ELISA, but not gp120 monomers or cleaved gp41. Two mAbs bound gp140 by mass-spectrometry and western blotting. Analysis of CDRH3 of the anti-HIV antibodies had a CDRH3 containing Cys and aromatic residues and were 14–22 amino acids (average size of 18.8 ± 3.1). In addition, the somatic mutation rate in CDRH3 compared to the DH3 germline gene was 82.35- 90.90% (Average: 87.34 ± 2.51%). The V-region sequence aligned most strongly with 2F5 and 4E10 patient derived mAbs.
Conclusions: The bovine V-gene CDRH3 size and frequency of somatic mutations of the isolated bovine Ig-genes raised through vaccination were comparable with those for human patient's elite neutralizing antibodies.
