Biology subjects

Gottumukkala, S.

Publications and source records attributed to Gottumukkala, S..

4 recordsLinked to original sources

bioRxiv2024

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

microbiology↗

bioRxiv2023

Structure-function Analyses Reveal Key Molecular Determinants of HIV-1 CRF01_AE Resistance to the Entry Inhibitor Temsavir

The HIV-1 entry inhibitor temsavir prevents CD4 from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir-resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveal that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad-antiviral activity.

molecular biology↗

bioRxiv2021

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

microbiology↗

bioRxiv2021

Across functional boundaries: making non-neutralizing antibodies to neutralize HIV-1 and mediate Fc-mediated effector killing of infected cells

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or Cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, closed Env trimer present on HIV-1 virions or expressed on HIV-1 infected cells. Antibodies targeting this region are therefore non-neutralizing and their potential as mediators of antibody depended cellular cytoxicity (ADCC) of HIV-1 infected cells diminished by a lack of available binding targets. Here we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or Cluster A specificity to the extracellular domain 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening closed Env trimers on HIV-1 particles/infected cells to expose the Cluster A region and activate ADCC and neutralization against these non-neutralizing targets. ImportanceHighly conserved epitopes within the co-receptor binding site (CoRBS) and constant region 1 and 2 (C1C2 or Cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4, therefore they are not accessible on virions and infected cells where the expression of CD4 is downregulated. Here we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or Cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1 infected cells through antibody depended cellular cytoxicity (ADCC) and inactivate HIV-1 virus through neutralization.

immunology↗