Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses

Abstract

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

Broadly resistant HIV-1 against CD4-binding site neutralizing antibodies

“…Major mechanisms appear to include a reduction of antibody binding to the contact residues and increased steric obstruction imposed by the bulky side-chains or glycansat the mutated residues.As the HIV-1 pandemic continues to evolve and particularly more CD4bs antibodies are entering clinical studies, more resistant strains are expected to arise. To overcome this resistance for optimal clinical outcomes, more potent and broadly effective antibodies are needed, either by isolating antibodies with exceptional neutralizing activity from patients,or engineering existing antibodies into bi-or tri-specific combinations [7,8,27,28,[65][66][67][68].A combination of VRC01 class and CDR H3-dominated antibodies, such as VRC13, may also be a beneficial alternative for developing therapeutic antibody cocktails due to their different sensitivity to resistance mutations at the CD4bs.…”

“…Sensitive strains are those susceptible to at least VRC01, 3BNC117, N6 and VRC13 with an IC 50 of less than 50 μg/ml.Based on these criteria, we identified a total of 19 broadly resistant strains and referred them as "Resistant panel to CD4bs bnAbs",including the nine selected here for more thorough analysis (CNE6, CNE23, CNE63, CNE64, CNE66, BJOX2000, BL01, TV1.29 and TZA125.17). On the other hand, we found113 sensitive strains with diverse genetic and geographic backgrounds [17,18,27,28].Sequence alignment of these 19 sequences against the standard sensitive strains HXB2, SF162 and JRFL revealed substantial differences of the CD4bs between the resistant and sensitive strains( Fig 1A).Most notable were changes in the inner domain, Loop D and β23/loop V5/β24 regions, which are critical for recognition by CD4bs bnAbs [17,18,[29][30][31]. Resistant strains also had alterations in potential Nglycosylation sites, particularly in the β23/loop V5/β24 region ( Fig 1A).We use the program WebLogo [51] to quantify altered residues in the epitope sequence that were disproportionally represented among the 19 resistant and 113 sensitive strains identified in the database ( Fig 1B).…”

“…Despite the superior potency and breadth of these CD4bs bnAbs, each fails to neutralize a small but significant portion of pseudotyped virus panels [17,25,27,28,37,38].For instance, VRC01 is unable to neutralize about 10% of tested viruses [25], and resistant strains have been isolated from patients infected with subtype B, subtype C, CRF07_BC, and CRF08_BC [29,[39][40][41][42]. Furthermore, reports show thatVRC01-, 3BNC117-and N6-resistant strains have emerged in both animal models and human clinical trials [11,15,[43][44][45][46][47], indicating that resistant virus Broadly resistant HIV-1 against CD4bs antibodies PLOS Pathogens | https://doi.org/10.…”

“…To identify residues potentially conferring broad resistance, we conducted comparative sequence analysis of resistant and sensitive strains available through the HIV Sequence Database (https://www.hiv.lanl.gov/) and published literature [17,18,27,28].Here,broad resistance is defined as a strain resistant to at least 10 of the 16 CD4bs bnAbs tested with an IC 50 of more than 50 μg/ml. Sensitive strains are those susceptible to at least VRC01, 3BNC117, N6 and VRC13 with an IC 50 of less than 50 μg/ml.Based on these criteria, we identified a total of 19 broadly resistant strains and referred them as "Resistant panel to CD4bs bnAbs",including the nine selected here for more thorough analysis (CNE6, CNE23, CNE63, CNE64, CNE66, BJOX2000, BL01, TV1.29 and TZA125.17).…”

“…The more recently isolated CD4bs bnAbs, such as 3BNC117, N6, N49P7, 3BNC60, VRC-PG04, VRC-PG20, NIH45-46, VRC-CH31, 12A12, CH103, 8ANC131, VRC13, and VRC16, have shown similar or even enhanced potency and breadth compared with VRC01 [1-5, 27, 28].The CD4bs bnAbs are classified into two groups based on their mode of recognition and heavy chain characteristics, such as the VRC01 class(3BNC117, N6, N49P7, 3BNC60, VRC-PG04, VRC-PG20, NIH45-46, VRC-CH31 and 12A12) and non-VRC01 classes (CH103, 8ANC131, VRC13 and VRC16) [17,18]. Structurally, these bnAbs recognize residues within the inner domain, the Loop D, the CD4 binding loop, and the β23/loop V5/β24 region [17,18,[29][30][31].The V1V2 and V3 loop also impact recognition,particularly in the context of the quaternary trimeric envelope protein [32][33][34][35][36].The primary mechanism of neutralization mimics and competes with the CD4 receptorto bind HIV-1 gp120, thereby preventing bound trimers from transitioning to the subsequent steps required for membrane fusion, although evidence indicates that there are differences in the fine details of this process [17,18,35,36].Despite the superior potency and breadth of these CD4bs bnAbs, each fails to neutralize a small but significant portion of pseudotyped virus panels [17,25,27,28,37,38].For instance, VRC01 is unable to neutralize about 10% of tested viruses [25], and resistant strains have been isolated from patients infected with subtype B, subtype C, CRF07_BC, and CRF08_BC [29,[39][40][41][42]. Furthermore, reports show thatVRC01-, 3BNC117-and N6-resistant strains have emerged in both animal models and human clinical trials [11,15,[43][44][45]…”