Background:
Broadly neutralizing monoclonal antibodies (bNAbs) target sites of vulnerability on the HIV envelope, such as the gp41 membrane proximal external region (MPER). MPER-directed bNAbs, such as VRC42.01 and PGZL1, have been previously isolated from chronically subtype CRF_01 AE and subtype B infected donors, respectively, and are part of the 4E10-class of MPER-specific bNAbs. Here we report the isolation of MHRP.01 from a subtype B infected donor, as another 4E10-class bNAb. These data indicate that MPER-directed mAbs are found in multiple donors infected with different HIV subtypes.
Methods:
Monoclonal antibodies (mAbs) were isolated from a subtype B infected donor using two parallel
Methods:
high-throughput B-cell culture followed by microneutralization assays, and fluorescently labeled MPER peptide to capture antigen-specific B cells by cell sorting. B-cell receptors were sequenced, and heavy and light chain sequences were cloned into expression vectors and expressed in Expi293F cells. MAbs were then characterized for binding, epitope mapping, and neutralization across a panel of 208 diverse pseudoviruses (pSVs). MAbs with potent cross-neutralization were further characterized by crystal structure determination.
Results:
MPER-specific mAbs were isolated using both MPER-specific B-cells and probe negative B-cells, and confirmed by neutralization using an HIV-2 chimera pSV containing HIV-2 backbone with HIV-1 MPER inserted. Two mAbs, MHRP.01 and MHRP.02 were able to neutralize 100% and 97% of the pSVs in a panel of 208, respectively with an average potency < 1ug/mL. MHRP.01 utilized VH1-69 and VK3-20, the same gene usage as other MPER-specific mAbs VRC42, and PGZL1. MHRP01-GW was produced to include shared amino acid residues with VRC42, glycine (G) and tryptophan (W), and exhibited more robust binding to the MPER peptide but did not improve neutralization compared to MHRP.01. Crystallization of MHRP.01 revealed residues important for MPER binding, which bound in the same conformation as MPER-directed bNAbs 4E10 and VRC42 (Fig. 1).
Conclusions:
These data reveal that similar monoclonal antibodies targeting MPER have been isolated from multiple individuals infected with different HIV subtypes. These mAbs, VRC42.01 (subtype CRF01_AE), PGZL1 (B) and now MHRP.01 (B) have identical gene usage, targeted epitope, angle of binding, and neutralization profiles, suggesting that individuals from different demographic areas could elicit B-cell clones targeting this epitope through vaccination efforts.
Structural comparisons of MHRP.01 with 4E10 and VRC42