Biology subjects

Prakash, S.

Publications and source records attributed to Prakash, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A Broad-Spectrum Multi-Antigen mRNA/LNP-Based Pan-Coronavirus Vaccine Induced Potent Cross-Protective Immunity Against Infection and Disease Caused by Highly Pathogenic and Heavily Spike-Mutated SARS-CoV-2 Variants of Concern in the Syrian Hamster Model

The first-generation Spike-alone-based COVID-19 vaccines have successfully contributed to reducing the risk of hospitalization, serious illness, and death caused by SARS-CoV-2 infections. However, waning immunity induced by these vaccines failed to prevent immune escape by many variants of concern (VOCs) that emerged from 2020 to 2024, resulting in a prolonged COVID-19 pandemic. We hypothesize that a next-generation Coronavirus (CoV) vaccine incorporating highly conserved non-Spike SARS-CoV-2 antigens would confer stronger and broader cross-protective immunity against multiple VOCs. In the present study, we identified ten non-Spike antigens that are highly conserved in 8.7 million SARS-CoV-2 strains, twenty-one VOCs, SARS-CoV, MERS-CoV, Common Cold CoVs, and animal CoVs. Seven of the 10 antigens were preferentially recognized by CD8+ and CD4+ T-cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC infection. Three out of the seven conserved non-Spike T cell antigens belong to the early expressed Replication and Transcription Complex (RTC) region, when administered to the golden Syrian hamsters, in combination with Spike, as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA/LNP-based pan-CoV vaccine): (i) Induced high frequencies of lung-resident antigen-specific CXCR5+CD4+ T follicular helper (TFH) cells, GzmB+CD4+ and GzmB+CD8+ cytotoxic T cells (TCYT), and CD69+IFN-{gamma}+TNF+CD4+ and CD69+IFN-{gamma}+TNF+CD8+ effector T cells (TEFF); and (ii) Reduced viral load and COVID-19-like symptoms caused by various VOCs, including the highly pathogenic B.1.617.2 Delta variant and the highly transmittable heavily Spike-mutated XBB1.5 Omicron sub-variant. The combined mRNA/LNP-based pan-CoV vaccine could be rapidly adapted for clinical use to confer broader cross-protective immunity against emerging highly mutated and pathogenic VOCs. IMPORTANCEAs of January 2024, over 1500 individuals in the United States alone are still dying from COVID-19 each week despite the implementation of first-generation Spike-alone-based COVID-19 vaccines. The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs), such as the currently circulating highly mutated BA.2.86 and JN.1 Omicron sub-variants, constantly overrode immunity induced by the first-generation Spike-alone-based COVID-19 vaccines. Here we report a next generation broad spectrum combined multi-antigen mRNA/LNP-based pan-CoV vaccine that consists of nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) that delivers three highly conserved non-Spike viral T cell protein antigens together with the Spike protein B-cell antigen. Compared side-by-side to the clinically proven first-generation Spike-alone mRNA/LNP-based vaccine, the combined multi-antigen mRNA/LNP-based pan-CoV vaccine-induced higher frequencies of lung-resident non-Spike antigen-specific T follicular helper (TFH) cells, cytotoxic T cells (TCYT), effector T cells (TEFF) and Spike specific-neutralizing antibodies. This was associated to a potent cross-reactive protection against various VOCs, including the highly pathogenic Delta variant and the highly transmittable heavily Spike-mutated Omicron sub-variants. Our findings suggest an alternative broad-spectrum pan-Coronavirus vaccine capable of (i) disrupting the current COVID-19 booster paradigm; (ii) outpacing the bivalent variant-adapted COVID-19 vaccines; and (iii) ending an apparent prolonged COVID-19 pandemic.

immunology↗

bioRxiv2023

Therapeutic Prime/Pull Vaccination of HSV-2 Infected Guinea Pigs with the Ribonucleotide Reductase 2 (RR2) Protein and CXCL11 Chemokine Boosts Antiviral Local Tissue-Resident and Effector Memory CD4+ and CD8+ T Cells and Protects Against Recurrent Genital Herpes

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes latency in sensory neurons of the dorsal root ganglia (DRG). Intermittent virus reactivation from latency and shedding in the vaginal mucosa (VM) causes recurrent genital herpes. While T-cells appear to play a role in controlling virus reactivation and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T-cells into DRG and VM tissues remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-1 RR2 protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 (AAV-8) expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus shedding in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ TRM and TEM cells in reducing virus reactivation shedding and the severity of recurrent genital herpes and propose the novel prime/pull vaccine strategy to protect against recurrent genital herpes. IMPORTANCEThe present study investigates a novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes in the guinea pig model. HSV-2 infected guinea pigs were vaccinated using a recombinantly expressed herpes tegument protein-RR2 (prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 (AAV-8) expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines (pull). The RR2/CXCL11 prime/pull therapeutic vaccine elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells in DRG and vaginal mucosa (VM) tissues in protection against recurrent genital herpes and propose the prime/pull therapeutic vaccine strategy in combating genital herpes. TWEETA therapeutic RR2/CXCL11 prime/pull vaccine reduced recurrent genital herpes more effectively than therapeutic vaccination with a subunit HSV RR2 antigen alone.

immunology↗

bioRxiv2023

Unequal genetic redundancy among the rice transcription factors OsMADS2 and OsMADS4 reveals distinct roles in floret organ development

Diversification of transcription factors and of their downstream targets can contribute to new organ morphologies. An example is rice lodicule, a small fleshy petal homolog that aids floret opening, thus facilitates pollination and fertility. To understand mechanisms underlying its specification, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2 and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2d8/d8 osmads4kd florets had severe abnormalities, were female infertile, yet initiated parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. Target genes whose regulation can contribute to OsMADS2 functions were discovered by its genome-wide binding analyses and transcriptome profiling. Several targets are implicated in lodicule and stamen development, floral organ number, cell wall, cell shape and osmotic homeostasis processes. Some targets relevant to lodicule development are cell division regulators (Cyclin D6, Cyclin P4-1-like), aquaporin (PIP1A), peptide transporter (PTR2), vascular development regulator (HOX1) and cell wall modulator (GH9B16). Their deregulation underscores the perturbed cell division, tissue differentiation patterns and physiology of the malformed osmads2 and osmads2d8/d8 osmads4kd lodicules. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion and embryo sac differentiation, divulge gene targets for lodicule development and provide mechanistic insights on the functional diversification of rice PI paralogs.

plant biology↗

bioRxiv2023

Cholesterol-associated Locus EHBP1 Protects against NASH Fibrosis

SUMMARYHepatic free cholesterol contributes to fibrosis in nonalcoholic steatohepatitis (NASH), but how hepatic cholesterol metabolism becomes dysregulated in NASH is not completely understood. We show here that human fibrotic NASH livers have decreased EHBP1, a novel GWAS locus associated with LDL-cholesterol, and that the EHBP1 rs10496099 T>C variant in NASH patients is associated with decreased hepatic EHBP1 expression and augmented NASH fibrosis. Congruent with the human data, EHBP1 loss- and gain-of-function increases and decreases NASH fibrosis in mice, respectively. Mechanistic studies reveal that EHBP1 promotes sortilin (SORT1)-mediated PCSK9 secretion, leading to LDLR degradation, decreased LDL uptake, and reduced TAZ, a fibrogenic effector. Moreover, we show that the TNF/PPAR pathway suppresses EHBP1 in NASH. These data not only provide new mechanistic insight into the role of EHBP1 in cholesterol metabolism and NASH fibrosis by uncovering the interaction between EHBP1 and other cholesterol-related loci, including SORT1, PCSK9, and LDLR, but also elucidate a novel interplay between inflammation and EHBP1-mediated cholesterol metabolism. HighlightsHepatic EHBP1 is reduced in human and mouse fibrotic NASH EHBP1 rs10496099 T>C is associated with decreased hepatic EHBP1 and augmented NASH EHBP1 reduces LDLR, cholesterol uptake, and TAZ in hepatocytes EHBP1 promotes sortilin-mediated PCSK9 secretion in hepatocytes TNF suppresses EHBP1 expression in hepatocytes

pathology↗

bioRxiv2023

Anti-Viral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein Against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models

SignificanceSARS-CoV-2 Variants of Concern (VOCs) continue to evolve and re-emerge with chronic inflammatory long-COVID sequelae necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the Receptor for Advanced Glycation End products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of the RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an anti-viral and anti-inflammatory therapeutic effect in the COVID-19 system. MethodsThe protective therapeutic effect of RAGE-Ig was determined in vitro in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six various VOCs of SARS-CoV-2. The underlying anti-viral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). ResultsFollowing treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated: (i) significant dose-dependent protection (i.e. greater survival, less weight loss, lower virus replication in the lungs); (ii) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (iii) a RAGE-Ig dose-dependent increase in the expression of type I interferons (IFN-, and IFN-{beta}) and type III interferon (IFN{lambda}2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (iv) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. ConclusionOur pre-clinical findings revealed type I and III interferons-mediated anti-viral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

immunology↗

bioRxiv2023

A Multi-Epitope/CXCL11 Prime/Pull Coronavirus Mucosal Vaccine Boosts the Frequency and the Function of Lung-Resident CD4+ and CD8+ Memory T Cells and Protects Against COVID-19-like Symptoms and Death Caused by SARS-CoV-2 infection

The pandemic of the coronavirus disease 2019 (COVID-19) has created the largest global health crisis in almost a century. Following exposure to SARS-CoV-2, the virus particles replicate in the lungs, induce a "cytokine storm" and potentially cause life-threatening inflammatory disease. Low frequencies of function SARS-CoV-2-specific CD4+ and CD8+ T cells in the lungs of COVID-19 patients were associated with severe cases of COVID-19. The apparent low level of T cell-attracting CXCL9, CXCL10, and CXCL11 chemokines in infected lungs may not be sufficient enough to assure the sequestration and/or homing of CD4+ and CD8+ T cells from the circulation into infected lungs. We hypothesize that a Coronavirus vaccine strategy that boosts the frequencies of functional SARS-CoV-2-specific CD4+ and CD8+ T cells in the lungs would lead to better protection against SARS-CoV-2 infection, COVID19-like symptoms, and death. In the present study, we designed and pre-clinically tested the safety, immunogenicity, and protective efficacy of a novel multi-epitope//CXCL11 prime/pull mucosal Coronavirus vaccine. This prime/pull vaccine strategy consists of intranasal delivery of a lung-tropic adeno-associated virus type 9 (AAV-9) vector that incorporates highly conserved human B, CD4+ CD8+ cell epitopes of SARS-CoV-2 (prime) and pulling the primed B and T cells into the lungs using the T cell attracting chemokine, CXCL-11 (pull). We demonstrated that immunization of HLA-DR*0101/HLA-A*0201/hACE2 triple transgenic mice with this multi-epitope//CXCL11 prime/pull Coronavirus mucosal vaccine: (i) Increased the frequencies of CD4+ and CD8+ TEM, TCM, and TRM cells in the lungs; and (ii) reduced COVID19-like symptoms, lowered virus replication, and prevented deaths following challenge with SARS-CoV-2. These findings discuss the importance of bolstering the number and function of lung-resident memory CD4+ and CD8+ T cells for better protection against SARS-CoV-2 infection, COVID-19-like symptoms, and death.

immunology↗

bioRxiv2023

High Frequencies of Antiviral Effector Memory TEM Cells and Memory B Cells Mobilized into Herpes Infected Vaginal Mucosa Associated With Protection Against Genital Herpes

Vaginal mucosa-resident anti-viral effector memory B- and T cells appeared to play a crucial role in protection against genital herpes. However, how to mobilize such protective immune cells into the vaginal tissue close to infected epithelial cells remains to be determined. In the present study, we investigate whether and how, CCL28, a major mucosal-associated chemokine, mobilizes effector memory B- and T cells in leading to protecting mucosal surfaces from herpes infection and disease. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). We found the presence of significant frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10 receptor, in herpes-infected asymptomatic (ASYMP) women compared to symptomatic (SYMP) women. A significant amount of the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP B6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected asymptomatic mice. In contrast, compared to wild-type (WT) B6 mice, the CCL28 knockout (CCL28(-/-)) mice: (i) Appeared more susceptible to intravaginal infection and re-infection with HSV-2; (ii) Exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. The results imply a critical role of the CCL28/CCR10 chemokine axis in the mobilization of anti-viral memory B and T cells within the VM to protect against genital herpes infection and disease.

immunology↗

bioRxiv2023

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗

bioRxiv2023

Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

Intracellular Salmonella resides and multiplies in cholesterol-rich specialized compartment called Salmonella-containing vacuoles (SCVs) and avoids fusion with acidic lysosomes. Given, lysosomes are primary organelle that redistributes LDL derived cholesterol to other organelles; we questioned how lysosomal cholesterol can be transported to SCV. We demonstrate here that peroxisomes are recruited to SCVs in human primary macrophages, epithelial cells and functions as pro-bacterial organelles. Further, this interaction is assisted by SseI, a Salmonella effector protein containing mammalian peroxisome targeting sequence. SseI localizes to peroxisome, interacts and activates a host Ras GTPase, ARF-1 on the peroxisome membrane. Activation of ARF-1 leads to recruitment of phosphatidylinsolitol-5- phosphate-4 kinase to generate phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. Accordingly, the{Delta} sseI strain showed reduced virulence in cell lines and during mice infection. Taken together, our work identified a fascinating mechanism by which a pathogen targets host organelles via its secretory effectors and exploits host metabolic intermediates for its intracellular proliferation.

cell biology↗

bioRxiv2023

DeepHeme: A generalizable, bone marrow classifierwith hematopathologist-level performance

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

bioinformatics↗

bioRxiv2023

Constitutive protein degradation induces acute cell death via proteolysis products

Modulation of proteolysis is an emerging therapeutic mainstay. The clinical success of thalidomide and analogs has inspired development of rationally-designed therapeutics that repurpose endogenous degradation machinery to target pathogenic proteins. However, it is unknown whether target removal is the critical effect that drives degrader-induced efficacy. Here we report that proteasome-generated peptides actively initiate degrader-induced cell death. Utilizing BET family degraders as exemplars, we find that induced proteasomal degradation of the BRD4-long isoform (BRD4-L) generates neo-amino-terminal peptides that neutralize Inhibitor of Apoptosis (IAP) proteins to precipitate cell death. Depletion of BRD4-L paradoxically suppresses caspase activation induced by numerous BET degraders. An unbiased screen revealed that other degrader compounds, including clinical CELMoDs, rely on the same mechanism to potentiate caspase activation and apoptosis. Finally, in the context of constitutive immunoglobulin proteostasis within multiple myeloma cells, we report that therapeutic proteasomal protease inhibition alters the peptide repertoire to neutralize IAPs, thus contributing to the clinical efficacy of bortezomib. Together, these findings clarify the counterintuitive clinical benefit achieved by combining thalidomide analogs with proteasome inhibitors. Our study reveals a previously unrealized pro-apoptotic function of the peptides generated by a variety of proteolysis-modulating compounds, that provide design considerations to maximize therapeutic benefit.

cell biology↗

bioRxiv2022

High Frequencies of Phenotypically and Functionally Senescent and Exhausted CD56+CD57+PD-1+ Natural Killer Cells, SARS-CoV-2-Specific Memory CD4+ and CD8+ T cells Associated with Severe Disease in Unvaccinated COVID-19 Patients

Unvaccinated COVID-19 patients display a large spectrum of symptoms, ranging from asymptomatic to severe symptoms, the latter even causing death. Distinct Natural killer (NK) and CD4+ and CD8+ T cells immune responses are generated in COVID-19 patients. However, the phenotype and functional characteristics of NK cells and T-cells associated with COVID-19 pathogenesis versus protection remain to be elucidated. In this study, we compared the phenotype and function of NK cells SARS-CoV-2-specific CD4+ and CD8+ T cells in unvaccinated symptomatic (SYMP) and unvaccinated asymptomatic (ASYMP) COVID-19 patients. The expression of senescent CD57 marker, CD45RA/CCR7differentiation status, exhaustion PD-1 marker, activation of HLA-DR, and CD38 markers were assessed on NK and T cells from SARS-CoV-2 positive SYMP patients, ASYMP patients, and Healthy Donors (HD) using multicolor flow cytometry. We detected significant increases in the expression levels of both exhaustion and senescence markers on NK and T cells from SYMP patients compared to ASYMP patients and HD controls. In SYMP COVID-19 patients, the T cell compartment displays several alterations involving naive, central memory, effector memory, and terminally differentiated T cells. The senescence CD57 marker was highly expressed on CD8+ TEM cells and CD8+ TEMRA cells. Moreover, we detected significant increases in the levels of proinflammatory TNF-, IFN-{gamma}, IL-6, IL-8, and IL-17 cytokines from SYMP COVID-19 patients, compared to ASYMP COVID-19 patients and HD controls. The findings suggest exhaustion and senescence in both NK and T cell compartment is associated with severe disease in critically ill COVID-19 patients. IMPORTANCEUnvaccinated COVID-19 patients display a large spectrum of symptoms, ranging from asymptomatic to severe symptoms, the latter even causing death. Distinct Natural killer (NK) and CD4+ and CD8+ T cells immune responses are generated in COVID-19 patients. In this study, we detected significant increases in the expression levels of both exhaustion and senescence markers on NK and T cells from unvaccinated symptomatic (SYMP) compared to unvaccinated asymptomatic (ASYMP) COVID-19 patients. Moreover, we detected significant increases in the levels of proinflammatory TNF-, IFN-{gamma}, IL-6, IL-8, and IL-17 cytokines from SYMP COVID-19 patients, compared to ASYMP COVID-19 patients. The findings suggest exhaustion and senescence in both NK and T cell compartment is associated with severe disease in critically ill COVID-19 patients. TWEETSignificant exhaustion and senescence in both NK and T cells were detected in unvaccinated symptomatic COVID-19 patients, suggesting a weakness in both innate and adaptive immune systems leads to severe disease in critically ill COVID-19 patients.

immunology↗

bioRxiv2022

A Prime/Pull RR2/CXCL11 Therapeutic Vaccine that Bolsters the Number and Function of Dorsal Root Ganglia Tissue-Resident HSV-Specific CD8+ TRM Cells Protects Latently Infected Guinea Pigs from Recurrent Genital Herpes

Reactivation of herpes simplex virus type 2 (HSV-2) from latently infected dorsal root ganglia (DRG) and subsequent virus shedding in the genital tract trigger recurrent genital herpes. Memory CD8+ T cells play a critical role in preventing HSV-2 reactivation from latently infected DRG, thus reducing recurrent genital lesions. The role of T-cell attracting chemokines in promoting CD8+ T cell protective immunity in recurrent genital herpes remains to be fully elucidated. In this study, we investigated whether and how the CXCL11/CXCR3 pathway affects the frequency and function of DRG-resident CD8+ T cells and the severity of recurrent genital herpes. Latently infected guinea pigs were primed with the HSV-1 RR2 protein, delivered intramuscularly with CpG/Alum adjuvants, and the induced T cells were "pulled" from the periphery into the latently infected DRG using T-cell attracting CXCL11 chemokine, delivered to DRG. In the guinea pigs that received the prime/pull vaccine, we detected a significant increase in both the number and function of tissue-resident IFN-{gamma}+CD103+CD44+CXCR3+CD8+ TRM cells that infiltrated healed sites of the vaginal mucosa (VM) and DRG tissues. This was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In contrast, in the guinea pigs that received the RR2 vaccine alone, we detected fewer functional CD8+ TRM cells and no reduction in the severity of recurrent genital herpes. These findings highlight the role of the CXCL11/CXCR3 chemokine pathway in shaping tissue-resident CD8+ TRM cell protective immunity against recurrent genital herpes. IMPORTANCERecurrent genital herpes is a common sexually transmitted disease worldwide. Currently, no FDA-approved therapeutic vaccines are available. In the present study, we used HSV-2 latently infected guinea pig to investigate a novel therapeutic prime/pull vaccine strategy based on priming T cells systemically, with a recombinantly expressed herpes envelope and tegument protein RR2 and "pulling" primed T cells into the tissues of latently infected ganglia with the T-cell-attracting chemokine, CXCL11. We discovered that this RR2/CXCL11 prime/pull vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with increased numbers of functional tissue-resident IFN-{gamma}+CD103+CD44+CXCR3+CD8+ TRM cells that infiltrate healed sites of the VM tissues and DRG. Our findings shed light on the role of TRM cells in protection against recurrent genital herpes and propose the prime/pull therapeutic vaccine as a new strategy against genital herpes. TWEETThe present study presents a novel RR2/CXCL11 prime/pull therapeutic vaccine that elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores.

immunology↗

bioRxiv2022

Quantification of genetic variants in bacterial cultures by Sanger sequencing

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify non-neutral mutations by selection or counter- selection, neutral mutations usually require DNA sequencing to be identified in a population, which are normally expensive and time-consuming. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counter- selection. We propose a novel methodology to quantify DNA using Sanger sequencing, that we validated experimentally with specially-engineered plasmids both in vitro and in co-transformed E. coli by and assessing our predictions with qPCR and fluorescence quantifications. The method relies on the alignment of the electropherograms from the query and reference samples, where we quantify the DNA concentration from the amplitude ratio of aligned electropherogram peaks. Our DNA quantification will allow quantifying genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with consistent reduction of costs compared to canonical approaches such as qPCR.

synthetic biology↗

bioRxiv2022

Engineered gene circuits with reinforcement learning allow bacteria to master gameplaying

Gene circuits enable cells to make decisions by controlling the expression of genes in reaction to specific environmental factors1. These circuits can be designed to encode logical operations2-7, but implementation of more complex algorithms has proved more challenging. Directed evolution optimizes gene circuits8 without the need for design knowledge9, but adjusting multiple genes and conditions10 in genotype searches poses challenges11. Here we show a multicellular sensor system, AdaptoCells, in Escherichia coli, that can evolve complex behavior through an accelerated adaptation to chemical environments. AdaptoCells recognize chemical patterns and act as a decision-making system. Using an iterative improvement method, we demonstrate that the AdaptoCells can evolve to achieve mastery in the game of tic-tac-toe, demonstrating an unprecedented level of complexity for engineered living cells. We provide an effective and straightforward way to encode complexity in gene circuits, allowing for fast adaptation in response to dynamic environments and leading to optimal decisions.

synthetic biology↗

bioRxiv2022

High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with -CCCs and {beta}-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-{gamma}-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with {beta}-CCCs strains but significantly fewer co-infections with -CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with -CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with -CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines. KEY POINTSO_LIA broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients. C_LIO_LIHigh frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients. C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens. C_LIO_LIThe cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients. C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the -CCCs strains. C_LIO_LICompared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the {beta}-CCCs strains. C_LI

immunology↗

bioRxiv2021

Antiviral CD19+CD27+ Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpes Infection

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4+ Tfh cells), that help B cells produce antibodies, were compared between HSV-1 infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that: (i) the ASYMP individuals had increased frequencies of HSV-specific CD19+CD27+ memory B cells; and (ii) high frequencies of HSV-specific switched IgG+CD19+CD27+ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0+CXCR5+CD4+ memory Tfh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD+CD138+ B cells within the TG and circulation of ASYMP mice compared to SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCEReactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice there was increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory Tfh cells. Moreover, following UV-B induce reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice, compared to SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

immunology↗

bioRxiv2021

Synthesis and characterization of peptide conjugated human serum albumin nanoparticles for targeted cardiac uptake and drug delivery.

Congestive heart failure, a prominent cardiovascular disease results primarily from myocardial infarction or ischemia. Milrinone (MRN), a widely used clinical drug for heart failure, improves myocardial contractility and cardiac function through its inotropic and vasodilatory effects. However, lacking target specificty, it exhibits low bioavailability and lower body retention time. Therefore, in this study, angiotensin II (AT1) peptide conjugated human serum albumin nanoparticles (AT1-HSA-MRN-NPs) have been synthesized for targeted delivery of MRN to the myocardium, overexpressing AT1 receptors under heart failure. The NPs were surface functionalized through a covalent conjugation reaction between HSA and AT1. Nanoparticle size was 215.2{+/-}4.7 nm and zeta potential -28.8{+/-}2.7 mV and cumulative release of MRN was [~]72% over 24 hrs. The intracellular uptake of nanoparticles and cell viability was studied in H9c2 cells treated with AT1-MRN-HSA-NPs vs the control non-targeted drug, MRN Lactate under normal, hypoxic and hypertrophic conditions. The uptake of AT1-HSA-MRN-NPs in H9c2 cells was significantly higher as compared to non-targeted nanoparticles, and the viability of H9c2 cells treated with AT1-MRN-HSA-NPs vs MRN Lactate was 73.4{+/-}1.4% vs 44.9{+/-}1.4%, respectively. Therefore, AT1-HSA-MRN-NPs are safe for in vivo use and exhibit superior targeting and drug delivery characteristics for treatment of heart failure.

bioengineering↗