Biology subjects

Mulligan, M. J.

Publications and source records attributed to Mulligan, M. J..

14 recordsLinked to original sources

bioRxiv2025

Persistent Classical and Atypical Memory B Cells Underlie Heterogeneous Vaccine Responses in Ocrelizumab-Treated Multiple Sclerosis

Background and ObjectivesPatients with multiple sclerosis (pwMS) treated with ocrelizumab (OCR), a B-cell-depleting therapy, exhibit heterogeneous humoral responses to SARS-CoV-2 mRNA vaccination. The mechanisms underlying this variability remain incompletely understood. We performed a longitudinal analysis of B-cell subset dynamics and antigen-specific T cell responses in OCR-treated pwMS and healthy controls to determine how immune cell composition, timing of OCR infusion, and lymphocyte dynamics influence humoral response outcomes. MethodsBased on post-vaccination anti-Spike IgG titers measured by multiplex bead immunoassay, pwMS were categorized as super-responders (SR), responders (R), or non-responders (NR). A 35-marker spectral flow cytometry panel was used to characterize T- and B-cell subsets longitudinally, at baseline and following stimulation with a SARS-CoV-2 peptide pool. ResultsCD4+ and CD8+ T cell populations were preserved across OCR-treated pwMS, and SARS-CoV-2-specific T cells remained detectable for more than 6 months after vaccination. In contrast, residual B-cell subset composition distinguished responders from non-responders. DN2-like B cells (CD19+CD27-IgD-T-bet+CD11c+CXCR5-) persisted despite repeated OCR infusions and were enriched in SR compared with NR. Repletion of mature naive B cells in peripheral blood correlated with time since last OCR infusion and with stronger humoral immune responses. ConclusionsB-cell subsets that resist OCR-mediated depletion may contribute to vaccine responsiveness in OCR-treated pwMS. Altered repletion kinetics of naive B cell subsets in non-responders suggest that specific B-cell populations may serve as predictive biomarkers of vaccine-induced humoral immunity, despite preserved T-cell responses.

immunology↗

bioRxiv2023

Multimodal characterization of antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection.

The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8+ T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8+ populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.

genomics↗

bioRxiv2022

Molecularly distinct memory CD4+ T cells are induced by SARS-CoV-2 infection and mRNA vaccination

Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history. One Sentence SummarySARS-CoV-2 infection versus SARS-CoV-2 mRNA vaccination prime durable transcriptionally and epigenetically distinct Spike-specific CD4+ T cell memory landscapes.

immunology↗

bioRxiv2022

The Vacc-SeqQC Project: Benchmarking RNA-Seq for Clinical Vaccine Studies

Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. RNA-Seq technology has matured in recent years and is now widely deployed for transcriptional analysis of clinical specimens. The goal of this study was to benchmark technical parameters of RNA-Seq in the context of a multi-site, double-blind randomized clinical trial using primary patient samples. We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two large-scale replicate datasets. We evaluated the impact of: (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data, and established the impact of read depth and gene filtering on transcript representation. Lastly, we modeled statistical power to detect DE genes for a range of sample sizes, effect sizes, and coverage depths. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

systems biology↗

bioRxiv2021

Increased resistance of SARS-CoV-2 Omicron Variant to Neutralization by Vaccine-Elicited and Therapeutic Antibodies

Currently authorized vaccines for SARS-CoV-2 have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant poses an obstacle both to vaccine protection and monoclonal antibody therapies. Analysis of the neutralization of Omicron spike protein-pseudotyped lentiviruses showed a 26-fold relative resistance (compared to D614G) to neutralization by convalescent sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titers against Omicron by 6-8-fold. Previous SARS-CoV-2 infection followed by vaccination resulted in the highest neutralizing titers against Omicron. Regeneron REGN10933 and REGN10987, and Lilly LY-CoV555 and LY-CoV016 monoclonal antibodies were ineffective against Omicron, while Sotrovimab was partially effective. The results highlight the benefit of a booster immunization in providing protection against Omicron but demonstrate the challenge to monoclonal antibody therapies.

immunology↗

bioRxiv2021

Neutralization of Mu and C.1.2 SARS-CoV-2 Variants by Vaccine-elicited Antibodies in Individuals With and Without Previous History of Infection

Recently identified SARS-CoV-2 variants Mu and C.1.2 have mutations in the receptor binding domain and N- and C-terminal domains that might confer resistance to natural and vaccine-elicited antibody. Analysis with pseudotyped lentiviruses showed that viruses with the Mu and C.1.2 spike proteins were partially resistant to neutralization by antibodies in convalescent sera and those elicited by mRNA and adenoviral vector-based vaccine-elicited antibodies. Virus with the C.1.2 variant spike, which is heavily mutated, was more neutralization-resistant than that of any of variants of concern. The resistance of the C.1.2 spike was caused by a combination of the RBD mutations N501Y, Y449H and E484K and the NTD mutations. Although Mu and C.1.2 were partially resistant to neutralizing antibody, neutralizing titers elicited by mRNA vaccination remained above what is found in convalescent sera and thus are likely to remain protective against severe disease. The neutralizing titers of sera from infection-experienced BNT162b2-vaccinated individuals, those with a history of previous SARS-CoV-2 infection, were as much as 15-fold higher than those of vaccinated individuals without previous infection and effectively neutralized all of the variants. The findings demonstrate that individuals can raise a broadly neutralizing humoral response by generating a polyclonal response to multiple spike protein epitopes that should protect against current and future variants.

immunology↗

bioRxiv2021

Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

immunology↗

bioRxiv2021

Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

immunology↗

bioRxiv2021

SARS-CoV-2 Lambda Variant Remains Susceptible to Neutralization by mRNA Vaccine-elicited Antibodies and Convalescent Serum

The SARS-CoV-2 lambda variant (lineage C.37) was designated by the World Health Organization as a variant of interest and is currently increasing in prevalence in South American and other countries. The lambda spike protein contains novel mutations within the receptor binding domain (L452Q and F490S) that may contribute to its increased transmissibility and could result in susceptibility to re-infection or a reduction in protection provided by current vaccines. In this study, the infectivity and susceptibility of viruses with the lambda variant spike protein to neutralization by convalescent sera and vaccine-elicited antibodies was tested. Virus with the lambda spike had higher infectivity and was neutralized by convalescent sera and vaccine-elicited antibodies with a relatively minor 2.3-3.3-fold decrease in titer on average. The virus was neutralized by the Regeneron therapeutic monoclonal antibody cocktail with no loss of titer. The results suggest that vaccines in current use will remain protective against the lambda variant and that monoclonal antibody therapy will remain effective.

immunology↗

bioRxiv2021

The Spike Proteins of SARS-CoV-2 B.1.617 and B.1.618 Variants Identified in India Provide Partial Resistance to Vaccine-elicited and Therapeutic Monoclonal Antibodies.

Highly transmissible SARS-CoV-2 variants recently identified in India designated B.1.617 and B.1.618 have mutations within the spike protein that may contribute to their increased transmissibility and that could potentially result in re-infection or resistance to vaccine-elicited antibody. B.1.617 encodes a spike protein with mutations L452R, E484Q, D614G and P681R while the B.1.618 spike has mutations {Delta}145-146, E484K and D614G. We generated lentiviruses pseudotyped by the variant proteins and determined their resistance to neutralization by convalescent sera, vaccine-elicited antibodies and therapeutic monoclonal antibodies. Viruses with B.1.617 and B.1.618 spike were neutralized with a 2-5-fold decrease in titer by convalescent sera and vaccine-elicited antibodies. The E484Q and E484K versions were neutralized with a 2-4-fold decrease in titer. Virus with the B.1.617 spike protein was neutralized with a 4.7-fold decrease in titer by the Regeneron monoclonal antibody cocktail as a result of the L452R mutation. The modest neutralization resistance of the variant spike proteins to vaccine elicited antibody suggests that current vaccines will remain protective against the B.1.617 and B.1.618 variants.

immunology↗

bioRxiv2021

Diversity and selection of SARS-CoV-2 minority variants in the early New York City outbreak

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. IMPORTANCEWhen viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

microbiology↗

bioRxiv2021

B.1.526 SARS-CoV-2 variants identified in New York City are neutralized by vaccine-elicited and therapeutic monoclonal antibodies

DNA sequence analysis recently identified the novel SARS-CoV-2 variant B.1.526 that is spreading at an alarming rate in the New York City area. Two versions of the variant were identified, both with the prevalent D614G mutation in the spike protein together with four novel point mutations and with an E484K or S477N mutation in the receptor binding domain, raising concerns of possible resistance to vaccine-elicited and therapeutic antibodies. We report that convalescent sera and vaccine-elicited antibodies retain full neutralizing titer against the S477N B.1.526 variant and neutralize the E484K version with a modest 3.5-fold decrease in titer as compared to D614G. The E484K version was neutralized with a 12-fold decrease in titer by the REGN10933 monoclonal antibody but the combination cocktail with REGN10987 was fully active. The findings suggest that current vaccines and therapeutic monoclonal antibodies will remain protective against the B.1.526 variants. The findings further support the value of wide-spread vaccination.

immunology↗

bioRxiv2021

Neutralization of viruses with European, South African, and United States SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies

The increasing prevalence of SARS-CoV-2 variants with mutations in the spike protein has raised concerns that recovered individuals may not be protected from reinfection and that current vaccines will become less effective. The B.1.1.7 isolate identified in the United Kingdom and B.1.351 isolate identified in the Republic of South Africa encode spike proteins with multiple mutations in the S1 and S2 subunits. In addition, variants have been identified in Columbus, Ohio (COH.20G/677H), Europe (20A.EU2) and in domesticated minks. Analysis by antibody neutralization of pseudotyped viruses showed that convalescent sera from patients infected prior to the emergence of the variant viruses neutralized viruses with the B.1.1.7, B.1.351, COH.20G/677H Columbus Ohio, 20A.EU2 Europe and mink cluster 5 spike proteins with only a minor decrease in titer compared to that of the earlier D614G spike protein. Serum specimens from individuals vaccinated with the BNT162b2 mRNA vaccine neutralized D614G virus with titers that were on average 7-fold greater than convalescent sera. Vaccine elicited antibodies neutralized virus with the B.1.1.7 spike protein with titers similar to D614G virus and neutralized virus with the B.1.351 spike with, on average, a 3-fold reduction in titer (1:500), a titer that was still higher than the average titer with which convalescent sera neutralized D614G (1:139). The reduction in titer was attributable to the E484K mutation in the RBD. The B.1.1.7 and B.1.351 viruses were not more infectious than D614G on ACE2.293T cells in vitro but N501Y, an ACE2 contacting residue present in the B.1.1.7, B.1.351 and COH.20G/677H spike proteins caused higher affinity binding to ACE2, likely contributing to their increased transmissibility. These findings suggest that antibodies elicited by primary infection and by the BNT162b2 mRNA vaccine are likely to maintain protective efficacy against B.1.1.7 and most other variants but that the partial resistance of virus with the B.1.351 spike protein could render some individuals less well protected, supporting a rationale for the development of modified vaccines containing E484K.

microbiology↗

bioRxiv2020

High titers of multiple antibody isotypes against the SARS-CoV-2 spike receptor-binding domain and nucleoprotein associate with better neutralization.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.

microbiology↗