Biology subjects

Ackerman, M. E.

Publications and source records attributed to Ackerman, M. E..

25 records · Page 2Linked to original sources

bioRxiv2023

Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2

Characterization of functional antibody responses to the N-terminal domain (NTD) of the SARS-CoV-2 spike (S) protein has included identification of both potent neutralizing activity and putative enhancement of infection. Fc{gamma}-receptor (Fc{gamma}R)-independent enhancement of SARS-CoV-2 infection mediated by NTD-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is not clear. Here we studied 1,213 S-binding mAbs derived from longitudinal sampling of B-cells collected from eight COVID-19 convalescent patients and identified 72 (5.9%) mAbs that enhanced infection in a VSV-SARS-CoV-2-S-Wuhan pseudovirus (PV) assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least five months post-infection. Enhancement of PV infection by NTD-binding mAbs was not observed using intestinal (Caco-2) and respiratory (Calu-3) epithelial cells as infection targets and was diminished or lost against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire from two of the convalescent subjects identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum (i.e., functionally secreted antibody). Functional analysis of these mAbs demonstrated robust activation of Fc{gamma}RIIIa associated with antibody binding to recombinant S proteins. Taken together, these findings suggest functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display diverse attributes that include Fc{gamma}R activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

immunology↗

bioRxiv2023

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that [~]50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower ([~]8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individuals total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

immunology↗

bioRxiv2023

Antibody effector functions are required for broad and potent protection of neonates from herpes simplex virus infection

The failure of multiple herpes simplex virus (HSV) vaccine candidates that induce neutralizing antibody responses raises the hypothesis that other activities, such as Fc domain-dependent effector functions, may be critical for protection. While neonatal HSV (nHSV) infection result in mortality and lifelong neurological morbidity in humans, it is uncommon among neonates with a seropositive birthing parent, suggesting the potential efficacy of antibody-based therapeutics to protect neonates. We therefore investigated the mechanisms of monoclonal antibody (mAb)-mediated protection in a mouse model of nHSV infection. Both neutralization and effector functions contributed to robust protection against nHSV-1. In contrast, effector functions alone were sufficient to protect against nHSV-2, exposing a functional dichotomy between virus types that is consistent with vaccine trial results. Together, these results emphasize that effector functions are crucial for optimal mAb-mediated protection, informing effective Ab and vaccine design, and demonstrating the potential of polyfunctional Abs as potent therapeutics for nHSV infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/555423v1_figa1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1045d4corg.highwire.dtl.DTLVardef@18946d9org.highwire.dtl.DTLVardef@18a5eaeorg.highwire.dtl.DTLVardef@14e1b54_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Mechanistic dissection of antibody-mediated protection from HSV.Monoclonal antibodies (mAbs) with varying neutralizing potencies and Fc modifications that impact effector function were evaluated in wildtype (WT) and Fc{gamma}R-/- mice to define mechanisms of antibody-mediated protection from HSV infection. To model human vulnerability to HSV disease during the neonatal period, neonatal mice were challenged with HSV, treated with mAb, and then assessed for morbidity and mortality. We observed that polyfunctional mAbs provide broader and more potent protection than antibodies with either low neutralization or low effector function. Moreover, while sufficient for protection against HSV-1, neutralization activity alone was unable to protect from HSV-2 infection. C_FIG

immunology↗

bioRxiv2023

Leveraging Permutation Testing to Assess Confidence in Positive-Unlabeled Learning Applied to High-Dimensional Biological Datasets

BackgroundCompared to traditional supervised machine learning approaches employing fully labeled samples, positive-unlabeled (PU) learning techniques aim to classify "unlabeled" samples based on a smaller proportion of known positive examples. This more challenging modeling goal reflects many real-world scenarios in which negative examples are not available, posing direct challenges to defining prediction accuracy robustness. While several studies have evaluated predictions learned from only definitive positive examples, few have investigated whether correct classification of a high proportion of known positives (KP) samples from among unlabeled samples can act as a surrogate to indicate a performance. ResultsIn this study, we report a novel methodology combining multiple established PU learning-based strategies to evaluate the potential of KP samples to accurately classify unlabeled samples without using "ground truth" positive and negative labels for validation. To address model robustness, we report the first application of permutation test in PU learning. Multivariate synthetic datasets and real-world high-dimensional benchmark datasets were employed to validate the proposed pipeline with varied underlying ground truth class label compositions among the unlabeled set and different proportions of KP examples. Comparisons between model performance with actual and permutated labels could be used to distinguish reliable from unreliable models. ConclusionsLike in fully supervised machine learning, permutation testing offers a means to set a baseline "no-information rate" benchmark in the context of semi-supervised PU learning inference tasks against which model performance can be compared.

bioinformatics↗

bioRxiv2022

Antibody-mediated prevention of vaginal HIV transmission is dictated by IgG subclass in humanized mice

HIV broadly neutralizing antibodies (bNAbs) are capable of both blocking viral entry and recruiting innate immunity to HIV-infected cells through their fragment crystallizable (Fc) region. Vaccination or productive infection results in a polyclonal mixture of class-switched IgG antibodies comprised of four subclasses, each encoding distinct Fc regions that differentially engage innate immune functions. Despite evidence that innate immunity contributes to protection, the relative contribution of individual IgG subclasses is unknown. Here we use vectored immunoprophylaxis (VIP) in humanized mice to interrogate the efficacy of individual IgG subclasses during prevention of vaginal HIV transmission by VRC07, a potent CD4-binding site directed bNAb. We find that VRC07-IgG2, which lacks Fc-mediated functionality, exhibits significantly reduced protection in vivo relative to other subclasses. However, even low concentrations of highly functional VRC07-IgG1 yields substantial protection against vaginal challenge, suggesting that interventions capable of eliciting modest titers of functional subclasses may provide meaningful benefit against infection.

immunology↗

bioRxiv2022

Maternally transferred monoclonal antibodies protect neonatal mice from herpes simplex virus-induced mortality and morbidity

Neonatal herpes simplex virus (HSV) infections often result in significant mortality and neurological morbidity despite antiviral drug therapy. Maternally-transferred HSV-specific antibodies reduce the risk of clinically-overt neonatal HSV (nHSV), but this observation has not been translationally applied. Using a neonatal mouse model, we tested the hypothesis that passive transfer of HSV-specific human monoclonal antibodies (mAbs) can prevent mortality and morbidity associated with nHSV. The mAbs were expressed in vivo by vectored immunoprophylaxis, or administered in vivo following recombinant expression in vitro. Through these maternally-derived routes or through direct administration to pups, diverse mAbs to HSV glycoprotein D protected against neonatal HSV-1 and HSV-2 infection. Using in vivo bioluminescent imaging, both pre- and post-exposure mAb treatment significantly reduced viral load. Administration of mAb also reduced nHSV-induced behavioral morbidity, as measured by anxiety-like behavior. Together these studies support the notion that HSV-specific mAb-based therapies may prevent or improve HSV infection outcomes in neonates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/476098v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@175f6eaorg.highwire.dtl.DTLVardef@1a59e39org.highwire.dtl.DTLVardef@36c484org.highwire.dtl.DTLVardef@19fec96_HPS_FORMAT_FIGEXP M_FIG C_FIG Different antibody sources were used to maternally-transfer or directly administer HSV-specific mAbs to mouse pups. Neonatal mice were challenged with wild type or bioluminescent virus before or after mAb acquisition. Following infection, pups were assessed for survival, virus-induced bioluminescence and anxiety-like behavior as a measure of neurological morbidity. Efficacy was time and mAb dependent. Notably, all HSV-specific mAbs prevented nHSV-associated mortality.

immunology↗

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↗