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1.
PLoS One ; 17(3): e0265236, 2022.
Article in English | MEDLINE | ID: covidwho-1938430

ABSTRACT

OBJECTIVE: To determine the seroprevalence of the SARS Cov 2 infection among vaccine naive population in a rural district of South India post-second surge. METHODOLOGY: We conducted a cross-sectional study in the five villages of a randomly chosen sub-district in the Bangalore rural district. We did house to house surveys and recruited 831 vaccine naive adults in July 2021. We tested samples for the presence of antibodies (including IgG & IgM) to SARS CoV-2 using the Roche Elecsys SARS-CoV-2 -S assay that quantifies antibodies against the receptor-binding domain (RBD) of the spike (S) protein. RESULTS: We estimated an overall prevalence of 62.7% (95% CI: 59.3-66.0) and an age-and gender-adjusted seroprevalence of 44.9% (95% CI: 42.5-47.4). When adjusted for test performance, the seroprevalence was 74.64% (95% CI: 70.66-78.47). The case-to-undetected-infected ratio (CIR) was 1: 8.65 (95% CI 1:8.1-1:9.1), and the Infection Fatality Rate (IFR) was 16.27 per 100,00 infections as of 13 July 2021. A history of at least one symptom suggestive of COVID-19 or a positive COVID-19 test of self or a family member in the past were significantly associated with seropositivity. CONCLUSION: We report a high seroprevalence of COVID-19 infection despite the advantages of low population density and well-ventilated landscapes in rural areas. CIR and IFR were higher than the previous serosurvey conducted in the same population during the first surge. The thought of achieving herd immunity comes with relief. However, it's vital to put efforts into building population health and rural health infrastructure to avert future health catastrophes.


Subject(s)
COVID-19/epidemiology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Antibodies, Viral/immunology , COVID-19 Vaccines , Cross-Sectional Studies , Female , Humans , Immunoglobulin G/immunology , India/epidemiology , Male , Middle Aged , Prevalence , RNA, Viral/immunology , Rural Population , SARS-CoV-2/pathogenicity , Seroepidemiologic Studies
2.
J Mol Biol ; 434(6): 167265, 2022 03 30.
Article in English | MEDLINE | ID: covidwho-1851575

ABSTRACT

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is characterized by a delayed interferon (IFN) response and high levels of proinflammatory cytokine expression. Type I and III IFNs serve as a first line of defense during acute viral infections and are readily antagonized by viruses to establish productive infection. A rapidly growing body of work has interrogated the mechanisms by which SARS-CoV-2 antagonizes both IFN induction and IFN signaling to establish productive infection. Here, we summarize these findings and discuss the molecular interactions that prevent viral RNA recognition, inhibit the induction of IFN gene expression, and block the response to IFN treatment. We also describe the mechanisms by which SARS-CoV-2 viral proteins promote host shutoff. A detailed understanding of the host-pathogen interactions that unbalance the IFN response is critical for the design and deployment of host-targeted therapeutics to manage COVID-19.


Subject(s)
COVID-19 , Immune Evasion , Interferons , SARS-CoV-2 , COVID-19/genetics , COVID-19/immunology , Gene Expression , Humans , Immunity, Innate , Interferons/genetics , RNA, Viral/immunology , SARS-CoV-2/immunology
3.
PLoS One ; 16(11): e0258819, 2021.
Article in English | MEDLINE | ID: covidwho-1706233

ABSTRACT

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.


Subject(s)
Antigens, Viral/immunology , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , COVID-19/virology , Humans , Limit of Detection , Point-of-Care Systems , RNA, Viral/immunology , Sensitivity and Specificity
5.
Genes (Basel) ; 13(1)2021 12 23.
Article in English | MEDLINE | ID: covidwho-1580896

ABSTRACT

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.


Subject(s)
Adenosine Deaminase/genetics , COVID-19/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , RNA Editing , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Adenosine/metabolism , Adenosine Deaminase/immunology , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Deamination , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Inosine/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-beta/genetics , Interferon-beta/immunology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Viral/immunology , RNA-Binding Proteins/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Transcriptome , Vero Cells
6.
J Clin Invest ; 131(23)2021 12 01.
Article in English | MEDLINE | ID: covidwho-1571525

ABSTRACT

Nucleoside-modified mRNA vaccines have gained global attention because of COVID-19. We evaluated a similar vaccine approach for preventing a chronic, latent genital infection rather than an acute respiratory infection. We used animal models to compare an HSV-2 trivalent nucleoside-modified mRNA vaccine with the same antigens prepared as proteins, with an emphasis on antigen-specific memory B cell responses and immune correlates of protection. In guinea pigs, serum neutralizing-antibody titers were higher at 1 month and declined far less by 8 months in mRNA- compared with protein-immunized animals. Both vaccines protected against death and genital lesions when infected 1 month after immunization; however, protection was more durable in the mRNA group compared with the protein group when infected after 8 months, an interval representing greater than 15% of the animal's lifespan. Serum and vaginal neutralizing-antibody titers correlated with protection against infection, as measured by genital lesions and vaginal virus titers 2 days after infection. In mice, the mRNA vaccine generated more antigen-specific memory B cells than the protein vaccine at early times after immunization that persisted for up to 1 year. High neutralizing titers and robust B cell immune memory likely explain the more durable protection by the HSV-2 mRNA vaccine.


Subject(s)
Herpes Genitalis , Herpesvirus 2, Human/immunology , Immunologic Memory , RNA, Viral/immunology , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Animals , COVID-19/immunology , COVID-19/prevention & control , Disease Models, Animal , Female , Guinea Pigs , Herpes Genitalis/immunology , Herpes Genitalis/prevention & control , SARS-CoV-2/immunology
7.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544295

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
8.
Expert Opin Ther Pat ; 31(12): 1177-1188, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1526144

ABSTRACT

Introduction: NGOs and governments of some countries have demanded suspension of patents protection of COVID-19 vaccines and the underlying technology to enhance worldwide access. At the same time, companies actually developing and producing COVID-19 vaccines have to navigate the patent landscape and have to deal with 3rd party patents.Areas covered: This article discusses these different aspects regarding patent protection of COVID-19 vaccines. Patent searches have been carried out in Espacenet and the ORBIT database. Different search strings were used by the author, based on his own background knowledge.Expert opinion: SARS-CoV 2 was for the first time fully described on 10 January 2020, so it is so far not possible to determine if, and by whom, patent applications were filed for respective vaccines. On that background, allegations that patents would be responsible for insufficient access to the vaccine in particular in developing countries are baseless. Even the key players are facing contraints caused by third-party patents, and legal disputes are already ongoing. Anyway, the bigger obstacle for worldwide equitable vaccine distribution seems to reside in know how transfer and production capacities, as ramping up production requires considerable efforts.


Subject(s)
COVID-19 Vaccines/therapeutic use , COVID-19/prevention & control , Patents as Topic , Humans , RNA, Viral/immunology
9.
Nat Med ; 27(11): 1990-2001, 2021 11.
Article in English | MEDLINE | ID: covidwho-1526094

ABSTRACT

SARS-CoV-2 messenger RNA vaccination in healthy individuals generates immune protection against COVID-19. However, little is known about SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses longitudinally in patients with multiple sclerosis (MS) on anti-CD20 antibody monotherapy (n = 20) compared with healthy controls (n = 10) after BNT162b2 or mRNA-1273 mRNA vaccination. Treatment with anti-CD20 monoclonal antibody (aCD20) significantly reduced spike-specific and receptor-binding domain (RBD)-specific antibody and memory B cell responses in most patients, an effect ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. By contrast, all patients with MS treated with aCD20 generated antigen-specific CD4 and CD8 T cell responses after vaccination. Treatment with aCD20 skewed responses, compromising circulating follicular helper T (TFH) cell responses and augmenting CD8 T cell induction, while preserving type 1 helper T (TH1) cell priming. Patients with MS treated with aCD20 lacking anti-RBD IgG had the most severe defect in circulating TFH responses and more robust CD8 T cell responses. These data define the nature of the SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making and public health policy for immunosuppressed patients including those treated with aCD20.


Subject(s)
COVID-19 Vaccines/therapeutic use , Multiple Sclerosis/immunology , Multiple Sclerosis/therapy , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/analysis , Antibodies, Viral/blood , Antigens, CD20/immunology , COVID-19/prevention & control , Case-Control Studies , Chlorocebus aethiops , HEK293 Cells , Humans , Immunity, Cellular , Immunity, Humoral/drug effects , Immunity, Humoral/physiology , Immunotherapy/methods , Longitudinal Studies , Multiple Sclerosis/blood , RNA, Messenger/immunology , RNA, Viral/immunology , Rituximab/pharmacology , Rituximab/therapeutic use , SARS-CoV-2/genetics , Vaccination , Vero Cells
10.
Curr Protein Pept Sci ; 22(4): 273-289, 2021 Oct 26.
Article in English | MEDLINE | ID: covidwho-1515505

ABSTRACT

Innate immunity is the first line of defence elicited by the host immune system to fight against invading pathogens such as viruses and bacteria. From this elementary immune response, the more complex antigen-specific adaptive responses are recruited to provide a long-lasting memory against the pathogens. Innate immunity gets activated when the host cell utilizes a diverse set of receptors known as pattern recognition receptors (PRR) to recognize the viruses that have penetrated the host and responds with cellular processes like complement system, phagocytosis, cytokine release and inflammation and destruction of NK cells. Viral RNA or DNA or viral intermediate products are recognized by receptors like toll-like receptors(TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) thereby, inducing type I interferon response (IFN) and other proinflammatory cytokines in infected cells or other immune cells. But certain viruses can evade the host innate immune response to replicate efficiently, triggering the spread of the viral infection. The present review describes the similarity in the mechanism chosen by viruses from different families -HIV, SARSCoV- 2 and Nipah viruses to evade the innate immune response and how efficiently they establish the infection in the host. The review also addresses the stages of developments of various vaccines against these viral diseases and the challenges encountered by the researchers during vaccine development.


Subject(s)
COVID-19/virology , HIV Infections/virology , Henipavirus Infections/virology , RNA, Viral/immunology , Viral Vaccines/immunology , Viruses , Animals , Humans , Immune Evasion , Immunity, Innate , Viruses/genetics , Viruses/immunology
11.
J Virol ; 96(2): e0167821, 2022 01 26.
Article in English | MEDLINE | ID: covidwho-1511415

ABSTRACT

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.


Subject(s)
COVID-19 , Host Microbial Interactions , RNA, Viral/immunology , SARS-CoV-2 , A549 Cells , COVID-19/immunology , COVID-19/virology , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Virus Internalization
12.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1481965

ABSTRACT

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.


Subject(s)
Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , RNA, Viral/administration & dosage , Replicon , Viral Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Defective Viruses/genetics , Defective Viruses/immunology , Female , Gene Deletion , Genes, env , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral/genetics , RNA, Viral/immunology , Vaccines, DNA , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics , Virulence/immunology
13.
Cell Mol Life Sci ; 78(23): 7427-7434, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1491059

ABSTRACT

Viral infections pose a severe threat to humans by causing many infectious, even fatal, diseases, such as the current pandemic disease (COVID-19) since 2019, and understanding how the host innate immune system recognizes viruses has become more important. Endosomal and cytosolic sensors can detect viral nucleic acids to induce type I interferon and proinflammatory cytokines, subsequently inducing interferon-stimulated genes for restricting viral infection. Although viral RNA and DNA sensing generally rely on diverse receptors and adaptors, the crosstalk between DNA and RNA sensing is gradually appreciated. This minireview highlights the overlap between the RNA- and DNA-sensing mechanisms in antiviral innate immunity, which significantly amplifies the antiviral innate responses to restrict viral infection and might be a potential novel target for preventing and treating viral diseases.


Subject(s)
COVID-19/immunology , DNA, Viral/immunology , Immunity, Innate/immunology , RNA, Viral/immunology , SARS-CoV-2/immunology , COVID-19/prevention & control , Cytokines/metabolism , Endosomes/immunology , Humans , Interferon Type I/metabolism , Membrane Proteins/immunology , Nuclear Proteins/immunology , Phosphoproteins/immunology
14.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1186: 123015, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1487818

ABSTRACT

The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.


Subject(s)
COVID-19 Vaccines/chemistry , Chromatography, Gel/methods , Drug Carriers/chemistry , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Vaccines/immunology , Drug Delivery Systems , Humans , Lipids/chemistry , Particle Size , RNA, Messenger/immunology , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology
15.
Acc Chem Res ; 54(21): 4012-4023, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1483069

ABSTRACT

In vitro-transcribed RNAs are emerging as new biologics for therapeutic innovation, as exemplified by their application recently in SARS-CoV-2 vaccinations. RNAs prepared by in vitro transcription (IVT) allow transient expression of proteins of interest, conferring safety over DNA- or virus-mediated gene delivery systems. However, in vitro-transcribed RNAs should be used with caution because of their immunogenicity, which is in part triggered by double-stranded RNA (dsRNA) byproducts during IVT. Cellular innate immune response to dsRNA byproducts can lead to undesirable consequences, including suppression of protein synthesis and cell death, which in turn can detrimentally impact the efficacy of mRNA therapy. Thus, it is critical to understand the nature of IVT byproducts and the mechanisms by which they trigger innate immune responses.Our lab has been investigating the mechanisms by which the innate immune system discriminates between "self" and "nonself" RNA, with the focus on the cytoplasmic dsRNA receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated 5 (MDA5). We have biochemically and structurally characterized critical events involving RNA discrimination and signal transduction by RIG-I or MDA5. We have used in vitro-transcribed RNAs as tools to investigate RNA specificity of RIG-I and MDA5, which required optimization of the IVT reaction and purification processes to eliminate the effect of IVT byproducts. In this Account, we summarize our current understanding of RIG-I and MDA5 and IVT reactions and propose future directions for improving IVT as a method to generate both research tools and therapeutics. Other critical proteins in cellular innate immune response to dsRNAs are also discussed. We arrange the contents in the following order: (i) innate immunity sensors for nonself RNA, including the RIG-I-like receptors (RLRs) in the cytosol and the toll-like receptors (TLRs) in the endosome, as well as cytoplasmic dsRNA-responding proteins, including protein kinase R (PKR) and 2',5'-oligoadenylate synthetases (OASes), illustrating the feature of protein-RNA binding and its consequences; (ii) the immunogenicity of IVT byproducts, specifically the generation of dsRNA molecules during IVT; and (iii) methods to reduce IVT RNA immunogenicity, including optimizations of RNA polymerases, reagents, and experimental conditions during IVT and subsequent purification.


Subject(s)
RNA, Viral/immunology , SARS-CoV-2/genetics , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , RNA, Viral/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/immunology
16.
Nat Med ; 27(11): 2002-2011, 2021 11.
Article in English | MEDLINE | ID: covidwho-1447313

ABSTRACT

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. In this study, we compared immune responses to the BNT162b2 mRNA Coronavirus Disease 2019 vaccine in patients with solid tumors (n = 53) who were on active cytotoxic anti-cancer therapy to a control cohort of participants without cancer (n = 50). Neutralizing antibodies were detected in 67% of patients with cancer after the first immunization, followed by a threefold increase in median titers after the second dose. Similar patterns were observed for spike protein-specific serum antibodies and T cells, but the magnitude of each of these responses was diminished relative to the control cohort. In most patients with cancer, we detected spike receptor-binding domain and other S1-specific memory B cell subsets as potential predictors of anamnestic responses to additional immunizations. We therefore initiated a phase 1 trial for 20 cancer cohort participants of a third vaccine dose of BNT162b2 ( NCT04936997 ); primary outcomes were immune responses, with a secondary outcome of safety. At 1 week after a third immunization, 16 participants demonstrated a median threefold increase in neutralizing antibody responses, but no improvement was observed in T cell responses. Adverse events were mild. These results suggest that a third dose of BNT162b2 is safe, improves humoral immunity against SARS-CoV-2 and could be immunologically beneficial for patients with cancer on active chemotherapy.


Subject(s)
/administration & dosage , COVID-19/prevention & control , Neoplasms/therapy , Adult , Aged , Antibodies, Viral/blood , Antibodies, Viral/metabolism , Arizona , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Cohort Studies , Dose-Response Relationship, Drug , Female , Humans , Immunity, Humoral/drug effects , Immunity, Humoral/physiology , Male , Middle Aged , Neoplasms/immunology , Neoplasms/pathology , RNA, Messenger/immunology , RNA, Viral/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Young Adult
17.
Virus Res ; 305: 198579, 2021 11.
Article in English | MEDLINE | ID: covidwho-1433887

ABSTRACT

The SARS-CoV2 mediated Covid-19 pandemic has impacted humankind at an unprecedented scale. While substantial research efforts have focused towards understanding the mechanisms of viral infection and developing vaccines/ therapeutics, factors affecting the susceptibility to SARS-CoV2 infection and manifestation of Covid-19 remain less explored. Given that the Human Leukocyte Antigen (HLA) system is known to vary among ethnic populations, it is likely to affect the recognition of the virus, and in turn, the susceptibility to Covid-19. To understand this, we used bioinformatic tools to probe all SARS-CoV2 peptides which could elicit T-cell response in humans. We also tried to answer the intriguing question of whether these potential epitopes were equally immunogenic across ethnicities, by studying the distribution of HLA alleles among different populations and their share of cognate epitopes. Results indicate that the immune recognition potential of SARS-CoV2 epitopes tend to vary between different ethnic groups. While the South Asians are likely to recognize higher number of CD8-specific epitopes, Europeans are likely to identify higher number of CD4-specific epitopes. We also hypothesize and provide clues that the newer mutations in SARS-CoV2 are unlikely to alter the T-cell mediated immunogenic responses among the studied ethnic populations. The work presented herein is expected to bolster our understanding of the pandemic, by providing insights into differential immunological response of ethnic populations to the virus as well as by gaging the possible effects of mutations in SARS-CoV2 on efficacy of potential epitope-based vaccines through evaluating ∼40,000 viral genomes.


Subject(s)
COVID-19/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Genome, Viral , HLA Antigens/immunology , SARS-CoV-2/immunology , Africa/epidemiology , Alleles , Amino Acid Sequence , Asia/epidemiology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , COVID-19/epidemiology , COVID-19/genetics , COVID-19/pathology , Computational Biology/methods , Disease Susceptibility , Epitopes, B-Lymphocyte/classification , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/classification , Epitopes, T-Lymphocyte/genetics , Europe/epidemiology , HLA Antigens/classification , HLA Antigens/genetics , Humans , Middle East/epidemiology , Oceania/epidemiology , Principal Component Analysis , RNA, Viral/genetics , RNA, Viral/immunology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
18.
Virus Res ; 305: 198555, 2021 11.
Article in English | MEDLINE | ID: covidwho-1412516

ABSTRACT

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Vaccines/chemistry , Propiolactone/pharmacology , SARS-CoV-2/drug effects , Virion/drug effects , Virus Inactivation/drug effects , Animals , Antigens, Viral/chemistry , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Flocculation/drug effects , Humans , Immune Sera/chemistry , RNA, Viral/chemistry , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Vaccines, Inactivated , Vero Cells , Virion/chemistry , Virion/immunology
19.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1406489

ABSTRACT

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Subject(s)
Algorithms , RNA, Double-Stranded/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Base Pairing , Base Sequence , COVID-19/prevention & control , Humans , Hydrolysis , RNA Stability , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Thermodynamics
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