Penrose, K. J. | Search bioRxiv

bioRxiv2026

Potent and Broad HIV-1 Neutralization by a Bispecific CD4-CD4i Fusion Protein based on Single-Domain CD4-D1 and X5 CD4i antibody

Human immunodeficiency virus (HIV) infection remains a global health threat. Although antiretroviral therapy (ART) has significantly transformed HIV into a manageable chronic disease, emergence of drug resistance to current ART is a continuing concern. Broadly neutralizing antibodies, as well as reagents containing both a soluble CD4 mimetic and an HIV co-receptor inhibitor--such as CD4-CD4i antibodies--are promising strategies for the prevention and treatment of HIV infection. We previously developed a CD4 D1mimetic (mD1.22) with enhanced neutralization potency, surpassing that of the clinically validated sCD4-D1D2 mimetic. We also previously identified a novel CD4i antibody (X5) that targets the conserved coreceptor binding site on the gp120 core and recognizes an epitope partially overlapping with 17b monoclonal antibody binding site. X5 binding to gp120 was augmented by CD4 and modestly enhanced by CCR5. To leverage these favorable interactions, we designed and optimized sCD4-X5 bispecific antibodies by computational structure-aided modification of antibody size and fusion linkers between the two binding moieties. The bispecific D1X5, with a (G4S)7 long linker between D1 and X5 (IgG1-LL D1X5), exhibited broad neutralization against 11 diverse HIV subtypes across B, C, G clades and AC, BC recombinants. The TZM-bl cell neutralization assay showed IgG1-LL D1X5 neutralization geometric mean IC50 and IC80 are 0.6 g/mL and 3.4 g/mL respectively, which are within the range of potent bnAbs. This work has identified a novel single domain soluble CD4 based CD4-CD4i bispecific antibody with broad HIV-1 neutralization.

molecular biology↗

bioRxiv2025

HIV-1 envelopes from virions that persist in plasma on antiretroviral therapy show reduced susceptibility to autologous immunoglobulins and variable sensitivity to broadly neutralizing monoclonal antibodies

Despite adherence to antiretroviral therapy (ART), low levels of plasma virus persist in most individuals with HIV-1. In a small subset of these individuals, this non-suppressible viremia is detected by routine clinical testing (plasma HIV-1 RNA >20 copies/ml) and has been shown to be of clonal cellular origin. The mechanisms by which these virions escape immune clearance is not defined, but reduced binding and neutralization of their surface envelope protein (Env) by antibodies (Abs) may contribute. To assess this possibility, we measured the monoclonal antibody (mAb) sensitivity of HIV-1 Env in plasma virus from 4 well-characterized individuals on ART with non-suppressible viremia. Thirty-two env plasma sequences were used to produce pseudovirus for neutralization assays with both autologous plasma and a panel of 15 recombinant antibodies that targeted different regions of HIV-1 Env protein. We found that autologous plasma had no neutralizing activity against the pseudoviruses consistent with persistence of viremia. In general, the Envs from the non-suppressible plasma virus were also less sensitive to mAb neutralization compared controls: tier 1 (6535), tier 2 (TRO11) or tier 3 (PVO) subtype B envs, although variability across Envs was evident. Two Env protein variants from one donor, R-09_A8 and R-09_C2, were less sensitive to VRC01 likely due to an additional N-glycan site at a VRC01 contact site and longer V5 regions. Most of the donor Envs were sensitive to at least two of three V3-glycan mAbs except for variant C-03_A6, which showed reduced sensitivity to all three. The CD4 binding site mAb 3BNC117 and the Gp41-specific mAb 10E8 neutralized pseudoviruses from all donors, indicating the potential for clearance of persistent viremia in these individuals studied. Author summaryIn a fraction of individuals on suppressive antiretroviral therapy (ART), episodes of persistent low-level viremia can be observed that is not attributed to ineffective ART and/or non-adherence. Previously in three individuals, we have shown that this viremia results from large HIV-1 infected T cell clones harboring replication-competent proviruses. The factor(s) that allow these infectious virions to be detected and not cleared by the immune system is unknown, suggesting that these virions can evade humoral responses. Using single genome sequencing, we amplified the env from plasma derived virions from these individuals, cloned env -containing amplicons into an expression vector to produce pseudo viruses, which we tested against either their autologous contemporaneous autologous immunoglobulins (Ig) or a panel of 15 recombinant monoclonal antibodies. The results revealed that these pseudovirus were not neutralized by their autologous Igs and exhibited complex pseudovirus-specific susceptibility profiles for the monoclonal antibodies they were tested against. Collectively, our findings suggest that despite resistance to autologous Ig, likely combinations of monoclonal antibodies will be needed to clear this persistent viremia.

immunology↗