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bioRxiv2020

Biochemical Patterns of Antibody Polyreactivity Revealed Through a Bioinformatics-Based Analysis of CDR Loops

Antibodies are critical components of adaptive immunity, binding with high affinity to pathogenic epitopes. Antibodies undergo rigorous selection to achieve this high affinity, yet some maintain an additional basal level of low affinity, broad reactivity to diverse epitopes, a phenomenon termed "polyreactivity". While polyreactivity has been observed in antibodies isolated from various immunological niches, the biophysical properties that allow for promiscuity in a protein selected for high affinity binding to a single target remain unclear. Using a database of nearly 1,500 polyreactive and non-polyreactive antibody sequences, we created a bioinformatic pipeline to isolate key determinants of polyreactivity. These determinants, which include an increase in inter-loop crosstalk and a propensity for an "inoffensive" binding surface, are sufficient to generate a classifier able to identify polyreactive antibodies with over 75% accuracy. The framework from which this classifier was built is generalizable, and represents a powerful, automated pipeline for future immune repertoire analysis.

immunology↗

bioRxiv2020

Identification of a prostaglandin E2 receptor that regulates mosquito oenocytoid immune cell function in limiting bacteria and parasite infection

Lipid-derived signaling molecules known as eicosanoids have integral roles in mediating immune and inflammatory processes across metazoans. This includes the function of prostaglandins and their cognate G protein-coupled receptors (GPCRs) to employ their immunological actions. In insects, prostaglandins have been implicated in the regulation of both cellular and humoral immune responses, yet studies have been limited by the absence of a described prostaglandin receptor. Here, we characterize a prostaglandin E2 receptor (AgPGE2R) in the mosquito Anopheles gambiae and examine its contributions to innate immunity. AgPGE2R expression is most abundant in circulating hemocytes where it is primarily localized to oenocytoid immune cell populations. Through the administration of prostaglandin E2 (PGE2) and AgPGE2R-silencing by RNAi, we demonstrate that PGE2 signaling regulates the expression of a subset of prophenoloxidases (PPOs) and antimicrobial peptides (AMPs). PGE2 priming via the AgPGE2R significantly limited bacterial replication and suppressed Plasmodium oocyst survival. Additional experiments establish that PGE2 priming increases phenoloxidase (PO) activity through the increased expression of PPO1 and PPO3, which significantly influence Plasmodium oocyst survival. We also provide evidence that PGE2 priming is concentration-dependent, where high concentrations of PGE2 promote oenocytoid lysis, negating the protective effects of PGE2 priming on anti-Plasmodium immunity. Taken together, our results characterize the AgPGE2R and the role of prostaglandin signaling on immune cell function, providing new insights into the role of PGE2 on anti-bacterial and anti-Plasmodium immune responses in the mosquito host.

immunology↗

bioRxiv2020

Immunosuppressive role of PGE2 during human tuberculosis

Prostaglandin E2 (PGE2), an active lipid compound derived from arachidonic acid, regulates different stages of the immune response of the host during several pathologies such as chronic infections or cancer. Manipulation of PGE2 levels was proposed as an approach for countering the Type I IFN signature of tuberculosis (TB), but very limited information exists about this pathway in patients with active TB. Here, we demonstrated that PGE2 exerts a potent immunosuppressive action during the immune response of the human host against M. tuberculosis. Thus, we showed that PGE2 inhibited both lymphoproliferation and cytokine production of proinflammatory cytokines, together with a significant reduction of the surface expression of several immunological receptors in human cells. However, PGE2 promoted the autophagic flux of antigen-stimulated monocytes, even in the presence of IFN. In this way, the attenuation of inflammation and immunopathology caused by an excessive immune response emerges as an attractive therapeutic target. Together, our findings contribute to the knowledge of Mtb-resistance mediated by PGE2 and highlight the potential of this lipid mediator as a tool to improve anti-TB treatment.

immunology↗

bioRxiv2020

CD40 provides immune privilege to the bone marrow hematopoietic niche

Allogeneic bone marrow transplantation remains the only therapeutic option for a wide range of hematological malignancies despite the risk of possible adverse, immune-related events, such as infection and acute graft-versus-host disease (aGVHD). aGVHD is characterized by T-cell activation, defective B-cell development and osteoblastic niche destruction in bone marrow (BM) among other issues. Transplant conditioning regimens cause excessive inflammatory cytokines production and impaired regulatory T-cell control of aberrant T-cell activation. Here, we show that mesenchymal cells (MSCs) upregulated CD40 upon irradiation at the expense of mesenchymal markers, and that CD40 endows MSC of regulatory function on Treg homeostasis and fitness. Transplantation of wild type hematopoietic cells into a CD40-null recipient reduces Treg numbers allowing persistent T-cell activation and pro-inflammatory cytokines production causing, impaired B-lymphopoiesis. These evidences find correlation in aGVHD patients showing the loss of CD40+ BM-MSCs along with reduction in cells of the B-lineage. Modeling aGVHD in mice we show that the elimination of CD40+ BM-MSCs relies on their higher expression of MHC-I molecules. Indeed, aGVHD mice compared to MHC-matched controls showed the loss of MHC-I + radio-resistant host BM-MSCs. Our data point to CD40+ MHC-I+BM-MSCs as a key regulator of BM tolerogenic niches. Key pointsO_LICD40 regulates BM immunological tolerance following total body irradiation (TBI) and transplantation (BMT). C_LIO_LILoss of CD40+MHC-IhighBM-MSCs is associated to BM manifestation of aGVHD in human and murine model. C_LI

immunology↗

bioRxiv2020

The identity of human tissue-emigrant CD8+ T cells

Lymphocyte migration is essential for human adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies to date have been restricted to immunological analyses of blood and various tissues. To address this issue, we used an integrated approach to characterize tissue-emigrant immune cells in thoracic duct lymph (TDL). In humans and non-human primates, lymphocytes were by far the most abundant immune lineage population in efferent lymph, and a vast majority of these lymphocytes were T cells. Cytolytic CD8+ T cell subsets were clonotypically discrete and selectively confined to the intravascular circulation, persisting for months after inhibition of S1P-dependent tissue egress by FTY-720. In contrast, non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Collectively, these data provide an atlas of the migratory immune system and define the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

immunology↗

bioRxiv2020

SARS-Cov-2-, HIV-1-, Ebola-neutralizing and anti-PD1 clones are predisposed

Antibody repertoire refers to the totality of the superbly diversified antibodies within an individual to cope with the vast array of possible pathogens. Despite this extreme diversity, antibodies of the same clonotype, namely public clones, have been discovered among individuals. Although some public clones could be explained by antibody convergence, public clones in naive repertoire or virus-neutralizing clones from not infected people were also discovered. All these findings indicated that public clones might not occur by random and they might exert essential functions. However, the frequencies and functions of public clones in a population have never been studied. Here, we integrated 2,449 Rep-seq datasets from 767 donors and discovered 5.07 million public clones - ~10% of the repertoire are public in population. We found 38 therapeutic clones out of 3,390 annotated public clones including anti-PD1 clones in healthy people. Moreover, we also revealed clones neutralizing SARS-CoV-2, Ebola, and HIV-1 viruses in healthy individuals. Our result demonstrated that these clones are predisposed in the human antibody repertoire and may exert critical functions during particular immunological stimuli and consequently benefit the donors. We also implemented RAPID - a Rep-seq Analysis Platform with Integrated Databases, which may serve as a useful tool for others in the field.

immunology↗

bioRxiv2020

Tissue-resident memory CD8 T-cell responses elicited by a single injection of a multi-target COVID-19 vaccine

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) which enters the body principally through the nasal and larynx mucosa and progress to the lungs through the respiratory tract. SARS-CoV-2 replicates efficiently in respiratory epithelial cells motivating the development of alternative and rapidly scalable vaccine inducing mucosal protective and long-lasting immunity. We have previously developed an immunologically optimized multi-neoepitopes-based peptide vaccine platform which has already demonstrated tolerance and efficacy in hundreds of lung cancer patients. Here, we present a multi-target CD8 T cell peptide COVID-19 vaccine design targeting several structural (S, M, N) and non-structural (NSPs) SARS-CoV-2 proteins with selected epitopes in conserved regions of the SARS-CoV-2 genome. We observed that a single subcutaneous injection of a serie of epitopes induces a robust immunogenicity in-vivo as measured by IFN{gamma} ELIspot. Upon tetramer characterization we found that this serie of epitopes induces a strong proportion of virus-specific CD8 T cells expressing CD103, CD44, CXCR3 and CD49a, the specific phenotype of tissue-resident memory T lymphocytes (Trm). Finally, we observed broad cellular responses, as characterized by IFN{gamma} production, upon restimulation with structural and non-structural protein-derived epitopes using blood T cells isolated from convalescent asymptomatic, moderate and severe COVID-19 patients. These data provide insights for further development of a second generation of COVID-19 vaccine focused on inducing lasting Th1-biased memory CD8 T cell sentinels protection using immunodominant epitopes naturally observed after SARS-CoV-2 infection resolution. Statement of SignificanceHumoral and cellular adaptive immunity are different and complementary immune defenses engaged by the body to clear viral infection. While neutralizing antibodies have the capacity to block virus binding to its entry receptor expressed on human cells, memory T lymphocytes have the capacity to eliminate infected cells and are required for viral clearance. However, viruses evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (phenomenon named antigenic drift). This limitation of the antibody-mediated immunity could be addressed by the T-cell mediated immunity, which is able to recognize conserved viral peptides from any viral proteins presented by virus-infected cells. Thus, by targeting several proteins and conserved regions on the genome of a virus, T-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. We designed a multi-target T cell-based vaccine containing epitope regions optimized for CD8+ T cell stimulation that would drive long-lasting cellular immunity with high specificity, avoiding undesired effects such as antibody-dependent enhancement (ADE) and antibody-induced macrophages hyperinflammation that could be observed in subjects with severe COVID-19. Our in-vivo results showed that a single injection of selected CD8 T cell epitopes induces memory viral-specific T-cell responses with a phenotype of tissue-resident memory T cells (Trm). Trm has attracted a growing interest for developing vaccination strategies since they act as immune sentinels in barrier tissue such as the respiratory tract and the lung. Because of their localization in tissues, they are able to immediately recognize infected cells and, because of their memory phenotypes, they rapidly respond to viral infection by orchestrating local protective immune responses to eliminate pathogens. Lastly, such multiepitope-based vaccination platform uses robust and well-validated synthetic peptide production technologies that can be rapidly manufactured in a distributed manner.

immunology↗

bioRxiv2020

The developing T-cell compartment of the neonatal lung orchestrates an atypical response to respiratory syncytial virus

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the specific features of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a state-of-the-art model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4 and CD8 T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while {gamma}{delta} T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity. AUTHOR SUMMARYBy using a state-of-the-art translational model with full accessibility to the small airways at defined early life periods, we provide an unpreceded characterization of the developing T cell compartment in the distal lungs of healthy and RSV-infected neonates. This process is highly dynamic and tightly regulated, characterized by colonizing T-cell subsets that synergize towards a narrow pro-tolerogenic immunological window. We believe our work constitutes a solid basis to clarify the age dependency of RSV immunopathogenesis, and should be considered in vaccine design, which remains challenging after five decades of effort.

immunology↗

bioRxiv2020

Lactobacillus rhamnosus attenuates bone loss and maintains bone health by skewing Treg-Th17 cell balance in Ovx mice

Osteoporosis is a systemic-skeletal disorder characterized by enhanced fragility of bones leading to increased rates of fractures and morbidity in large number of populations. Probiotics are known to be involved in management of various-inflammatory diseases including osteoporosis. But no study till date had delineated the immunomodulatory potential of Lactobacillus rhamnosus (LR) in bone-health. In the present study, we examine the effect of probiotic-LR on bone-health in osteoporotic (Ovx) mice model. We observed that administration of LR attenuated bone-loss in Ovx mice. Both the cortical and trabecular bone-content of LR treated group was significantly higher than Ovx-group. Remarkably, the percentage of osteoclastogenic-CD4+Ror{gamma}t+Th17 cells at distinct immunological sites such as BM, spleen, LN and PP were significantly reduced, whereas the percentage of anti-osteoclastogenic-CD4+Foxp3+Tregs and CD8+Foxp3+Tregs were significantly enhanced in LR-treated group thereby resulting in inhibition of bone-loss. The immunomodulatory-role of LR was further supported by serum-cytokine data with a significant reduction in proinflammatory-cytokines (IL-6, IL-17 and TNF-) along with enhancement in anti-inflammatory-cytokines (IL-10, IFN-{gamma}) in LR treated-group. Altogether, the present study for the first time establishes the osteoprotective role of LR on bone-health, thus highlighting the potential of LR in the treatment and management of various bone related diseases including osteoporosis.

immunology↗

bioRxiv2020

Epitopes targeted by T cells in convalescent COVID-19 patients

Knowledge of the epitopes of SARS-CoV-2 that are targeted by T cells in convalescent patients is important for understanding T cell immunity against COVID-19. This information can aid the design, development and assessment of COVID-19 vaccines, and inform novel diagnostic technologies. Here we provide a unified description and meta-analysis of emerging data of SARS-CoV-2 T cell epitopes compiled from 15 independent studies of cohorts of convalescent COVID-19 patients. Our analysis demonstrates the broad diversity of T cell epitopes that have been collectively recorded for SARS-CoV-2, while also identifying a selected set of immunoprevalent epitopes that induced consistent T cell responses in multiple cohorts and in a large fraction of tested patients. The landscape of SARS-CoV-2 T cell epitopes that we describe can help guide future immunological studies, including those related to vaccines and diagnostics. A web-based platform has been developed to help complement these efforts.

immunology↗

bioRxiv2020

Metformin enhances anti-mycobacterial responses by educating immunometabolic circuits of CD8+ T cells

Diabetic patients taking metformin have lower risk for Mycobacterium tuberculosis (Mtb) infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence. However, a detailed mechanistic understanding of metformins protective immunological benefits on host resistance to TB is lacking. In this study, using mass cytometry we show that metformin treatment expands memory-like antigen-inexperienced CD8+CXCR3+ T cells in naive mice, and in healthy and diabetic humans. Metformin-educated CD8+ T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8+ T cells from CXCR3-/- mice did not exhibit metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhanced immunogenicity and protective efficacy against Mtb challenge. Collectively, our results demonstrate an important role of CD8+ T cells in metformin-derived host metabolic-fitness towards Mtb infection.

immunology↗

bioRxiv2020

HIV-specific T-cell responses reflect substantive in vivo interactions with infected cells despite long-term therapy

Antiretroviral therapies (ART) durably suppress HIV replication to undetectable levels - however, infection persists in the form of long-lived reservoirs of infected cells with integrated proviruses, that re-seed systemic replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in driving reductions in viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle, by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. We show that T-cell responses to autologous reservoir viruses persist over years, and that the maintenance of HIV-Nef-specific responses was uniquely associated with residual frequencies of infected cells. These responses disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV-infected cells. These results indicate substantial visibility of the HIV reservoir to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing HIV infection.

immunology↗

bioRxiv2020

Loss of mitochondrial protease CLPP activates type I interferon responses through the mtDNA-cGAS-STING signaling axis

Caseinolytic mitochondrial matrix peptidase proteolytic subunit, CLPP, is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome (PS), but also display immune system alterations. However, the molecular mechanisms and signaling pathways underlying immunological changes in CLPP null mice remain unclear. Here we report the steady state activation of type I interferon (IFN-I) signaling and antiviral gene expression in CLPP deficient cells and tissues. Depletion of the cyclic GMP-AMP (cGAS)-Stimulator of Interferon Genes (STING) DNA sensing pathway ablates heightened IFN-I responses and abrogates the broad viral resistance phenotype of CLPP null cells. Moreover, we report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene expression, implicating mtDNA stress as the driver of IFN-I signaling in CLPP null mice. Our work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have implications for understanding myriad human diseases involving CLPP dysregulation.

immunology↗

bioRxiv2020

Optimal CD4T Cell Priming in Lymph Nodes Requires Repertoire Scanning by CD301b+ Migratory cDC2 Cells

Activation of CD4T cells by conventional dendritic cells (cDC) is pivotal in adaptive immunity. However, while the activation mechanism of antigen-specific CD4T cells has been extensively studied, the cellular mechanism that leads to the selection of cognate CD4T cell clones out of the polyclonal pool is incompletely understood. Here, we show that, in the reactive lymph nodes, newly homed naive polyclonal CD4T cells are temporarily retained before leaving the lymph node. This stop-and-go traffic of CD4T cells provides an adequate time window for efficient scanning and timely priming of antigen-specific clones. Mechanistically, upon immunization, CD301b+ DCs, a major subset of migratory cDC2 cells, quickly migrate to the draining lymph node and settle in the areas near the high endothelial venules, where they retain incoming polyclonal CD4T cells through MHCII-dependent but antigen-independent mechanisms while concurrently providing cognate stimuli to prime antigen-specific CD4T cells. These results indicate that CD301b+ DCs function as an immunological "display window" for CD4T cells to efficiently scan their antigen specificity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/276410v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@118d9b7org.highwire.dtl.DTLVardef@7daf2eorg.highwire.dtl.DTLVardef@17808c0org.highwire.dtl.DTLVardef@186780e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINewly homed polyclonal CD4T cells are temporarily retained in the reactive lymph nodes. C_LIO_LIDepletion of CD301b+ DCs results in shorter dwell time of CD4T cells in the draining lymph node and delayed priming of antigen-specific clones. C_LIO_LIThe transient retention of polyclonal CD4T cells in the draining lymph node requires MHCII expression on CD301b+ DCs but not cognate antigen. C_LIO_LICD301b+ DCs are required for robust expansion of rare antigen-specific CD4T cell clones and their skewing toward Th2 cells. C_LI

immunology↗

bioRxiv2020

Single-cell transcriptomic analyses define distinct peripheral B cell subsets and discrete development pathways

Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the "Double negative" (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterise cells based on their transcriptome. To maximise immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamical transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. One involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and on its own developmental branch but with links to the classical memory branch.

immunology↗

bioRxiv2020

Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BackgroundElucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. ResultsHere, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluate methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5 v1 and 3 v3 methods. We demonstrate that these methods have fewer drop-out events which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. ConclusionOverall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

immunology↗

bioRxiv2020

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch Vaccines designed by reverse epitomics approach, shows potential to cover large ethnically distributed human population worldwide

BackgroundThe SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a positive-sense single-stranded RNA coronavirus responsible for the ongoing 2019-2020 COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. In present study we have designed and theoretically validated novel Multi-Patch Vaccines against SARS-CoV-2. MethodologyA novel reverse epitomics approach, "overlapping-epitope-clusters-to-patches" method is utilized to identify multiple antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are here termed as "Ag-Patch or Ag-Patches", for Antigenic Patch or Patches. The identification of Ag-Patches is based on clusters of overlapping epitopes rising from a particular region of SARS-CoV-2 protein. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel methodology for vaccine design and development. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. ResultsWe identified 73 CTL (Cytotoxic T-Lymphocyte), 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 (518 CTL and 250 HTL) overlapping epitopes targeting different HLA alleles. Such large number of epitope coverage is not possible for multi-epitope vaccines. The large number of epitopes covered implies large number of HLA alleles targeted, and hence large ethnically distributed human population coverage. The MPVs:Toll-Like Receptor ectodomain complex shows stable nature with numerous hydrogen bond formation and acceptable root mean square deviation and fluctuation. Further, the cDNA analysis favors high expression of the MPVs constructs in human cell line. ConclusionHighly immunogenic novel Ag-Patches are identified from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach. We conclude that the novel Multi-Patch Vaccines could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/284992v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@176f27org.highwire.dtl.DTLVardef@82a4fcorg.highwire.dtl.DTLVardef@11db43forg.highwire.dtl.DTLVardef@12495b2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOABSTRACT FIGURE:C_FLOATNO A Multi-Patch Vaccine design to combat SARS-CoV-2 and a method to prepare thereof. Multi-Patch Vaccine designing to combat SARS-CoV-2 infection by reverse epitomics approach, "Overlapping-epitope-clusters-to-patches" method. C_FIG

immunology↗

bioRxiv2020

T cell competition profoundly reduces the effect of initial precursor frequency on the generation of CD4 T cell memory

Protective immune responses are accompanied by increases in the frequency of high affinity T cells, which contribute to subsequent immunological memory. There is evidence that the fold-change in T cell number during the immune response is inversely related to initial precursor frequency, but the size of this effect remains poorly defined. Indeed, in many reports precursor frequency has been considered as directly proportional to the magnitude of the response. We have determined the effect of initial precursor frequency over the course of an in vivo antigen-specific response, in an experimental setting in which the other variables, TCR affinity and antigen dose, are kept constant. A major effect of precursor frequency was apparent in both the expansion and contraction phases; low initial precursor frequency in the physiological range was associated with greater initial expansion in T cell numbers, and also with preferential retention of memory cells. The effect was seen continuously across a 1000-fold naive cell frequency range, leading to memory cell frequencies that differed by only 3-fold. These results are consistent with the existence of ongoing competition for antigen throughout the course of the immune response and explain the paradoxical ability of populations of genetically diverse individuals to make appropriate protective immune responses despite the large differences in initial repertoire that result from semi-random thymic TCR repertoire generation and selection.

immunology↗