Biology subjects

Sanders, R.

Publications and source records attributed to Sanders, R..

9 recordsLinked to original sources

bioRxiv2022

Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide maturation of other bnAb classes. To compare antigens designed to maintain this conformation, apex-specific responses were monitored in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

immunology↗

bioRxiv2021

Evaluation of plasma N-terminal pro-B-type natriuretic peptide levels in healthy North American Salukis with normal echocardiographic measurements.

Measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels has been shown to have clinical significance for diagnosis and management of heart disease in dogs. Evaluation of current reference limits for specific breeds is necessary to ensure the test can accurately distinguish between healthy and diseased animals. The objective of this study is to evaluate the adequacy of currently established NT-proBNP reference limits for clinical use in healthy Salukis. Cardiac health of 33 clinically healthy Salukis was evaluated via echocardiography using available breed standards. Plasma concentrations of NT-proBNP were measured using a commercially available assay. A one-sided 97.5 % upper reference limit for the NT-proBNP concentrations was calculated using non-parametric percentile method. The 97.5 % upper reference limit was 769 pmol/L (90% CI, 547-1214 pmol/L) for the study dogs. This upper reference limit was within the currently established non-breed specific NT-proBNP upper reference limit of 900 pmol/L. No relationship between sex, age, or body weight on plasma levels of NT-proBNP was noted. Results of this study supports the use of currently available non-breed specific NT-proBNP cut-off values for clinical evaluation of healthy Salukis.

physiology↗

bioRxiv2021

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4-0.5 [A]. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.

molecular biology↗

bioRxiv2021

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within [~]1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

microbiology↗

bioRxiv2021

High transgene expression is associated with systemic GFP silencing in Nicotiana benthamiana

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand systemic transgene silencing in Nicotiana benthamiana. Previous reports details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event revealed inadvertent co-integration of part of a bacterial transposase. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous 16C lines produced for this study ranged from 50-72% of the homozygous 16C line. GFP expression was equivalent to 16C in a two-copy event. Local GFP silencing was observed in all transgenic and 16C hemizygous lines after topical application of delivery formulations with a GFP targeting dsRNA. The 16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of systemic transgene silencing in N. benthamiana.

plant biology↗

bioRxiv2020

Anti-SARS-CoV-2 IgG from severely ill COVID-19 patients promotes macrophage hyper-inflammatory responses

For yet unknown reasons, severely ill COVID-19 patients often become critically ill around the time of activation of adaptive immunity. Here, we show that anti-Spike IgG from serum of severely ill COVID-19 patients induces a hyper-inflammatory response by human macrophages, which subsequently breaks pulmonary endothelial barrier integrity and induces microvascular thrombosis. The excessive inflammatory capacity of this anti-Spike IgG is related to glycosylation changes in the IgG Fc tail. Moreover, the hyper-inflammatory response induced by anti-Spike IgG can be specifically counteracted in vitro by use of the active component of fostamatinib, an FDA- and EMA-approved therapeutic small molecule inhibitor of Syk. One sentence summaryAnti-Spike IgG promotes hyper-inflammation.

immunology↗

bioRxiv2020

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

The rapid spread of SARS-CoV-2 has a significant impact on global health, travel and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated neutralizing antibodies from convalescent COVID-19 patients using a SARS-CoV-2 stabilized prefusion spike protein. Several of these antibodies were able to potently inhibit live SARS-CoV-2 infection at concentrations as low as 0.007 {micro}g/mL, making them the most potent human SARS-CoV-2 antibodies described to date. Mapping studies revealed that the SARS-CoV-2 spike protein contained multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as previously undefined non-RBD epitopes. In addition to providing guidance for vaccine design, these mAbs are promising candidates for treatment and prevention of COVID-19.

microbiology↗

bioRxiv2020

Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

22 nt miRNAs or siRNAs have been shown to specifically induce production of transitive (secondary) siRNAs for targeted mRNAs. An abrasion method to deliver dsRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for 3 endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GUN4. However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs, but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also had more transitive siRNAs produced in response to 22 nt siRNAs, but the response varied from weak (CHL-I) to strong (CHL-H). 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for GFP, CHL-H and GUN4 in N. benthamiana. The special activity of 22 nt siRNAs in producing a greater local phenotype and induction of elevated levels of transitive siRNAs was also shown in A. cruentus for the CHL-H gene. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

plant biology↗

bioRxiv2019

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a key phenotype observed in DiGeorge syndrome patients and are caused by a microdeletion on chromosome 22q11. Heterozygosity of TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key component for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9 deficient mice are born with complex cardiovascular malformations affecting the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared to Tbx1 heterozygous mice. Using a novel Pax9Cre allele we demonstrated that the site of this Tbx1-Pax9 genetic interaction is in the pharyngeal endoderm, therefore revealing that a Tbx1/Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for critical tissue interactions during normal morphogenesis of the pharyngeal arch artery system.\n\nSummary statementPax9 is required for outflow tract and aortic arch development, and functions together with Tbx1 in the pharyngeal endoderm for 4th arch artery formation.

developmental biology↗