Biology subjects

Cronin, K.

Publications and source records attributed to Cronin, K..

10 recordsLinked to original sources

bioRxiv2024

Anti-HIV-1 B cell antigen receptor signaling and structure

The B cell antigen receptor (BCR) complex, comprised of antigen recognition and signaling components, functions in initiating B cell activation. While structural studies have described BCR domain organization, gaps remain in our understanding of its antigen binding domain (Fab, fragment antigen-binding) disposition, and how antigen binding is sensed to initiate signaling. Here, we report antigen affinity and signaling of the immunoglobulin (Ig) class IgM and IgG BCRs and define conformational states of full-length BCRs of two human broadly neutralizing antibodies, the glycan-specific, heavy chain domain-swapped, I-shaped 2G12, and a canonical Y-shaped antibody, CH31, that recognizes the CD4-binding site on the HIV-1 Envelope protein (Env). The BCRs adopted the shapes (I or Y) of their respective soluble antibodies, and both Ig class of BCRs of the same specificity bound Env trimers with similar affinities. We observed antigen-valenc y dependent differential signaling by the 2G12 IgM and IgG BCRs with trimeric Envs. Cryo-electron microscopy of the 2G12 IgG and CH31 IgM BCRs revealed varied Fab orientations. Structural comparisons revealed hinge points and regions of flexibility in the BCRs suggesting a highly dynamic structure of the BCR complex. Taken together, our results provide an integrated understanding of BCR structure, conformation, antigen recognition and signaling, and provide the basis for understanding antigen induced BCR signal transmission. One Sentence SummaryCryo-electron microscopy structures of full-length B cell antigen receptor complex provide a novel dynamic BCR model and a basis for understanding antigen binding induced signal transmission.

immunology↗

bioRxiv2023

Class-specific sensing of HIV-1 antigens by the B cell antigen receptor depends on the CH1 domain

How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here we study antigen binding and sensing of Ramos B cells expressing BCRs of either the IgM or IgG1 class with a specificity for the CD4-binding-site of the envelope (Env) protein of the human immune deficiency virus-1 (HIV-1). We find that, in spite of their identical antigen binding site, the two BCR classes differ drastically from each other in that the IgM-BCR and IgG1-BCR bind preferentially to monovalent and polyvalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR we found that the class-specific antigen-sensing behavior can be transferred with the CH1{gamma} domain from the IgG1-BCR to the IgM-BCR. Our results indicate that the class-switching process not only results in the production of antibody classes with different effector functions but also alters the antigen sensing of secondary B lymphocytes. These findings suggest that antigen valency in existing vaccination protocols should be modified and altered between primary versus secondary (booster) immunization.

immunology↗

bioRxiv2023

Germline-targeting SOSIP trimer immunization elicits precursor CD4 binding-site targeting broadly neutralizing antibodies in infant macaques.

A vaccine that can achieve protective immunity prior to sexual debut is critical to prevent the estimated 410,000 new HIV infections that occur yearly in adolescents. As children living with HIV can make broadly neutralizing antibody (bnAb) responses in plasma at a faster rate than adults, early childhood is an opportune window for implementation of a multi-dose HIV immunization strategy to elicit protective immunity prior to adolescence. Therefore, the goal of our study was to assess the ability of a B cell lineage-designed HIV envelope SOSIP to induce bnAbs in early life. Infant rhesus macaques (RMs) received either BG505 SOSIP or the germline-targeting BG505 GT1.1 SOSIP (n=5/group) with the 3M-052-SE adjuvant at 0, 6, and 12 weeks of age. All infant RMs were then boosted with the BG505 SOSIP at weeks 26, 52 and 78, mimicking a pediatric immunization schedule of multiple vaccine boosts within the first two years of life. Both immunization strategies induced durable, high magnitude binding antibodies and plasma autologous virus neutralization that primarily targeted the CD4-binding site (CD4bs) or C3/465 epitope. Notably, three BG505 GT1.1-immunized infants exhibited a plasma HIV neutralization signature reflective of VRC01-like CD4bs bnAb precursor development and heterologous virus neutralization. Finally, infant RMs developed precursor bnAb responses at a similar frequency to that of adult RMs receiving a similar immunization strategy. Thus, a multi-dose immunization regimen with bnAb lineage designed SOSIPs is a promising strategy for inducing protective HIV bnAb responses in childhood prior to adolescence when sexual HIV exposure risk begins.

immunology↗

bioRxiv2022

Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

SARS-CoV-2 Omicron variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of antibodies induced by vaccination. Here, we describe the SARS-CoV-2 neutralizing SP1-77 antibody that was generated from a humanized mouse model with a single human VH1-2 and V{kappa}1-33-associated with immensely diverse complementarity-determining-region-3 (CDR3) sequences. SP1-77 potently and broadly neutralizes SARS-CoV-2 variants of concern and binds the SARS-CoV-2 spike protein receptor-binding-domain (RBD) via a novel-CDR3-based mode. SP1-77 does not block RBD-binding to the ACE2-receptor or endocytosis step of viral entry, but rather blocks membrane fusion. Our findings provide the first mechanistic insight into how a non-ACE2 blocking antibody potently neutralizes SARS-CoV-2, which may inform strategies for designing vaccines that robustly neutralize current and future SARS-CoV-2 variants.

immunology↗

bioRxiv2022

B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

immunology↗

bioRxiv2021

Different adjuvanted pediatric HIV envelope vaccines induced distinct plasma antibody responses despite similar B cell receptor repertoires in infant rhesus macaques.

Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein coadministered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires.

immunology↗

bioRxiv2021

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

immunology↗

bioRxiv2019

Microbial community dynamics of surface water in British Columbia, Canada

Traditional methods for monitoring the microbiological quality of water focus on the detection of fecal indicator bacteria such as Escherichia coli, often tested as a weekly grab sample. To understand the stability of E.coli concentrations over time, we evaluated three approaches to measuring E. coli levels in water: microbial culture using Colilert, quantitative PCR for uidA and next-generation sequencing of the 16S rRNA gene. Two watersheds, one impacted by agricultural and the other by urban activities, were repeatedly sampled over a simultaneous ten-hour period during each of the four seasons. Based on 16S rRNA gene deep sequencing, each watershed showed different microbial community profiles. The bacterial microbiomes varied with season, but less so within each 10-hour sampling period. Enterobacteriaceae comprised only a small fraction (<1%) of the total community. The qPCR assay detected significantly higher quantities of E. coli compared to the Colilert assay and there was also variability in the Colilert measurements compared to Health Canadas recommendations for recreational water quality. From the 16S data, other bacteria such as Prevotella and Bacteroides showed promise as alternative indicators of fecal contamination. A better understanding of temporal changes in watershed microbiomes will be important in assessing the utility of current biomarkers of fecal contamination, determining the best timing for sample collection, as well as searching for additional microbial indicators of the health of a watershed.

microbiology↗

bioRxiv2017

Real-time genomic and epidemiological investigation of a multi-institution outbreak of KPC-producing Enterobacteriaceae: a translational study

BackgroundUntil recently, KPC-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.\n\nMethods and FindingsAll KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing (WGS) was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids. Initial investigations indicated the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012-2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.\n\nConclusionsA real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase producing Enterobacteriaceae management.

genomics↗

bioRxiv2017

Antibiotic resistance genes in agriculture and urban influenced watersheds in southwestern British Columbia

BackgroundThe dissemination of antibiotic resistance genes (ARGs) from anthropogenic activities into the environment poses an emerging public health threat. Water constitutes a major vehicle for transport of both biological material and chemical substances. The present study focused on putative antibiotic resistance and integrase genes present in the microbiome of agricultural, urban influenced and protected watersheds in southwestern British Columbia, Canada. A metagenomics approach and high throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and integron-associated integrase genes (intI). Sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a one-year period.\n\nResultsData mining using CARD and Integrall databases enabled the identification of putative antibiotic resistance genes present in watershed samples. Antibiotic resistance genes presence in samples from various watershed locations was low relative to the microbial population (<1 %). Analysis of the metagenomic sequences detected a total of 78 ARGs and intI1 across all watershed locations. The relative abundance and richness of antibiotic resistance genes was found to be highest in agriculture impacted watersheds compared to protected and urban watersheds. Gene copy numbers (GCNs) from a subset of 21 different elements of antibiotic resistance were further estimated using HT qPCR. Most GCNs of ARGs were found to be variable over time. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Urban and agriculture impacted sites had a higher GCNs of ARGs compared to protected sites. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was negative. However, the high richness and GCNs of ARGs in impacted sites suggest effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the water column.\n\nConclusionAntibiotic resistance and integrase genes in a year-long metagenomic study showed that ARGs were driven mainly by environmental factors from anthropogenized sites in agriculture and urban watersheds. Environmental factors accounted for almost 40% of the variability observed in watershed locations.

microbiology↗