Biology subjects

Sullivan, N.

Publications and source records attributed to Sullivan, N..

4 recordsLinked to original sources

bioRxiv2021

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

immunology↗

bioRxiv2021

Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529

With B.1.1.529 SARS-CoV-2 variants rapid spread and substantially increased resistance to neutralization by vaccinee and convalescent sera, monoclonal antibodies with potent neutralization are eagerly sought. To provide insight into effective neutralization, we determined cryo-EM structures and evaluated potent receptor-binding domain (RBD) antibodies for their ability to bind and neutralize this new variant. B.1.1.529 RBD mutations altered 16% of the RBD surface, clustering on a ridge of this domain proximal to the ACE2-binding surface and reducing binding of most antibodies. Significant inhibitory activity was retained, however, by select monoclonal antibodies including A19-58.1, B1-182.1, COV2-2196, S2E12, A19-46.1, S309 and LY-CoV1404, which accommodated these changes and neutralized B.1.1.529 with IC50s between 5.1-281 ng/ml, and we identified combinations of antibodies with potent synergistic neutralization. Structure-function analyses delineated the impact of resistance mutations and revealed structural mechanisms for maintenance of potent neutralization against emerging variants. Summary SentenceWe show potent B.1.1.529 neutralization by select antibodies and use EM structures to reveal how potency can be retained.

immunology↗

bioRxiv2020

Activation of bone marrow adaptive immunity in type 2 diabetes: rescue by co-stimulation modulator Abatacept

Hypothesis Type 2 diabetes (T2D) is characterized by low-grade inflammation. Here, we investigated the state of adaptive immunity in bone marrow (BM) of patients and mice with T2D. We also tested if inhibition T cell co-stimulation by Abatacept could rescue the immune profile of T2D mice.Methods Flow-cytometry and cytokine analyses were performed on BM samples from patients with or without T2D. Moreover, we studied the immune profile of db/db and control wt/db mice. A cohort of db/db mice was randomized to receive Abatacept or vehicle for 4 weeks, with endpoints being immune cell profile and indexes of insulin sensitivity and heart performance.Results T2D patients showed increased frequencies of BM CD4+ (2.8-fold, p=0.001) and CD8+ T cells (1.8-fold, p=0.01), with upregulation of the activation marker CD69 and homing receptor CCR7 in CD4+ (1.64-fold, p=0.003 and 2.27-fold, p=0.01, respectively) and CD8+ fractions (1.79-fold, p=0.05 and 1.69-fold, p=0.02, respectively). CCL19 (CCR7 receptor ligand) and CXCL10/11 (CXCR3 receptor ligands), implicated in T cell migration and activation, were the most differentially modulated chemokines. Studies in mice confirmed the activation of adaptive immunity in T2D. Abatacept reduced the activation of T cells and levels of pro-inflammatory chemokines and cytokines. Additionally, Abatacept improved indexes of cardiac systolic function, but not insulin sensitivity.Conclusions These novel findings support the concept of BM adaptive immune activation in T2D. Modulation of T cell co-stimulation could represent an attractive and immediately available modality to dampen inappropriate activation of adaptive immune response and protect from target organ damage.Competing Interest StatementThe authors have declared no competing interest.Abbreviations(APCs)antigen-presenting cells(BM)bone marrow(BMMCs)BM mononuclear cells(CO)cardiac output(TCM)central memory(CCR7)CC-chemokine receptor 7(DCs)dendritic cells(EDV)end diastolic volume(TEM)effector memory(ND)non-diabetic control(HSPCs)hematopoietic stem/progenitor cells(SV)stroke volume(TEMRA)terminally differentiated T memory cells(T2D)type 2 diabetes mellitus(VAT)visceral adipose tissueView Full Text

immunology↗

bioRxiv2019

Extensive CD8β depletion does not prevent control of viral replication or protection from challenge in macaques chronically infected with a live attenuated simian immunodeficiency virus

We evaluated the contribution of CD8{beta}+ T cells on control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8-specific depleting monoclonal antibody (mAb), the CD8{beta}-specific mAb CD8{beta}255R1 selectively depleted CD8{beta}+ T cells without also depleting non-CD8+ T cell populations that express CD8, such as natural killer (NK) cells and {gamma}{delta} T cells. Following infusion with CD8{beta}255R1, plasma viremia transiently increased coincident with declining peripheral CD8{beta}+ T cells. Interestingly, plasma viremia returned to pre-depletion levels even when peripheral CD8{beta}+ T cells did not. Although depletion of CD8{beta}+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were three-fold lower (p=0.006) in animals that received CD8{beta}255R1 compared to control IgG. It is possible that these residual SIV-specific CD8{beta}+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8{beta}255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8{beta}255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8{beta}255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8{beta}255R1 infusion on the immunological composition of the host, when compared to an isotype matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8{beta}255R1 administration.\n\nImportanceStudies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8-specific mAbs that target both the major (CD8{beta}+) and minor (CD8+) populations of CD8+ T cells, but additionally deplete non-CD8+ T cell populations that express CD8, such as NK cells and {gamma}{delta} T cells. In the current study, we administered the CD8{beta}-specific depleting mAb CD8{beta}255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8{beta}+ T cells without removing CD8+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8{beta}255R1 for studying the direct contribution of CD8{beta}+ T cells in various disease states.

immunology↗